Methods for the treatment of interleukin-6 receptor-related diseases and disorders

ABSTRACT

The invention relates to amino acid sequences that are directed against and/or that can specifically bind to IL-6 receptor, compounds or constructs that comprise the amino acid sequence, nucleic acids that encode the amino acid sequences, compounds or constructs, pharmaceutical compositions comprising the amino acid sequences, compounds or constructs as well as methods for the prevention and/or treatment of diseases and disorders associated with IL-6 receptor.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/259,192, filed Apr. 23, 2014, now issued as U.S. Pat. No. 9,605,072,which is a divisional of U.S. application Ser. No. 14/143,022, filedDec. 30, 2013, now issued as U.S. Pat. No. 9,611,326, which is adivisional of U.S. application Ser. No. 12/310,223, filed Dec. 8, 2009,now issued as U.S. Pat. No. 8,629,244, which is national stage filingunder 35 U.S.C. § 371 of international application PCT/EP2007/058587,filed Aug. 17, 2007, which was published under PCT Article 21(2) inEnglish, and claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 60/838,904, filed Aug. 18, 2006, U.S.provisional application Ser. No. 60/873,012, filed Dec. 5, 2006, andU.S. provisional application Ser. No. 60/938,325, filed May 16, 2007,the disclosures of which are incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

The present invention relates to amino acid sequences that are directedagainst (as defined herein) Interleukin-6 Receptor (IL-6R), as well asto compounds or constructs, and in particular proteins and polypeptides,that comprise or essentially consist of one or more such amino acidsequences (also referred to herein as “amino acid sequences of theinvention”, “compounds of the invention”, and “polypeptides of theinvention”, respectively).

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

BACKGROUND OF THE INVENTION

The interaction of IL-6, a protein originally identified as a B celldifferentiation factor (Hirano et al., 1985; EP0257406), with IL-6R(Yamasaki et al., 1988; EP0325474) results in the formation of theIL-6/IL-6R complex. This complex binds to gp130 (Taga et al., 1989;EP0411946), a membrane protein on a target cell, which transmits variousphysiological actions of IL-6. IL-6 is currently known to be involvedin—amongst others—the regulation of the immune response, hematopoiesis,the acute phase response, bone metabolism, angiogenesis, andinflammation. Deregulation of IL-6 production is implicated in thepathology of several autoimmune and chronic inflammatory proliferativedisease processes (Ishihara and Hirano, 2002). As a consequence,inhibitors of IL-6 induced signaling have attracted much attention inthe past (Hirano et al., 1990). Polypeptides specifically binding toIL-6 (Klein et al., 1991; EP0312996), IL-6R (EP0409607) or gp130 (Saitoet al., 1993; EP0572118) proved to exhibit an efficient inhibitoryeffect on IL-6 functioning.

IL-6 overproduction and signalling (and in particular so-calledtrans-signalling) are involved in various diseases and disorders, suchas sepsis (Starnes et al., 1999) and various forms of cancer such asmultiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cellleukaemia (Klein et al., 1991), lymphoma, B-lymphoproliferative disorder(BLPD) and prostate cancer. Non-limiting examples of other diseasescaused by excessive IL-6 production or signalling include boneresorption (osteoporosis) (Roodman et al., 1992; Jilka et al., 1992),cachexia (Strassman et al., 1992), psoriasis, mesangial proliferativeglomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma (Emilie etal., 1994), inflammatory diseases and disorder such as rheumatoidarthritis, systemic onset juvenile idiopathic arthritis,hypergammaglobulinemia (Grau et al., 1990); Crohn's disease, ulcerativecolitis, systemic lupus erythematosus (SLE), multiple sclerosis,Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (inparticular allergic asthma) and autoimmune insulin-dependent diabetesmellitus (Campbell et al., 1991). Other IL-6 related disorders will beclear to the skilled person.

As can for example be seen from the references above, the prior artdescribes antibodies and antibody fragments directed against human IL-6,against human IL-6R and against human gp130 protein for the preventionand treatment of IL-6 relates disorders. Examples are Tocilizumab (seeWoo P, et al. Arthritis Res Ther. (2005) 7: 1281-8, Nishimoto N et al.Blood. (2005) 106: 2627-32, Ito H et al. Gastroenterology. (2004) 126:989-96, Choy E H et al. Arthritis Rheum. (2002) 46: 3143-50.), BE8 (seeBataille R et al. Blood (1995) 86:685-91, Emilie D et al. Blood (1994)84:2472-9, Beck J T et al. N Engl J Med. (1994) 330:602-5, Wendling D etal. J Rheumatol. (1993) 20:259-62.) and CNTO-328 of Centocor (seeJournal of Clinical Oncology, (2004) 22/14S: 2560; Journal of ClinicalOncology, (2004) 22/14S: 2608; Int J Cancer (2004) 111:592-5). Anotheractive principle known in the art for the prevention and treatment ofIL-6 related disorders is an Fc fusion of soluble gp130 (see Becker C etal. Immunity. (2004) 21: 491-501, Doganci A et al. J Clin Invest. (2005)115:313-25, Nowell M A et al. J Immunol. (2003) 171: 3202-9, Atreya R etal. Nat Med. (2000) 6:583-8.)

SUMMARY OF THE INVENTION

The amino acid sequences, polypeptides and compositions of the presentinvention can generally be used to modulate, and in particular inhibitand/or prevent, binding of IL-6R to IL-6 and/or binding of theIL-6/IL-6R complex to gp130 and thus to modulate, and in particularinhibit or prevent, the signalling that is mediated by IL-6R, IL-6,IL6/IL-6R complex or gp130 to modulate the biological pathways in whichIL-6R, IL-6, the IL6/IL-6R complex and/or gp130 are involved, and/or tomodulate the biological mechanisms, responses and effects associatedwith such signalling or these pathways.

In the context of the present invention “modulating the interactionbetween IL-6/IL-6R complex and gp130” can for example mean:

-   -   binding to IL-6R (i.e. as such or as present in the IL-6/IL-6R        complex) in such a way that the formation of the IL-6/IL-6R        complex is inhibited or affected (e.g. fully or partially        disrupted) in such a way that the binding of the complex to—e.g.        its affinity for—gp130 is reduced (or reversely, that the        binding of gp 130 to—e.g. its affinity for—the complex is        reduced), so that the signaling induced/mediated by the binding        of the complex to gp130 is modulated (e.g. reduced);        or    -   binding to IL-6R (i.e. as such or as present in the IL-6/IL-6R        complex) in such a way that the formation of the IL-6/IL-6R        complex essentially is not affected but that the binding of said        complex to gp130 is modulated (e.g. inhibited), so that the        signalling induced/mediated by the binding of the complex to        gp130 is modulated (e.g. reduced);        both compared to the formation of the complex and its binding to        gp130 without the presence of the amino acid sequence or        Nanobody of the invention.

Accordingly, in one specific, but non-limiting aspect, the inventionprovides polypeptides and compositions that are, and/or that can be usedas, an antagonist of IL-6, of IL-6R, of IL-6- or IL-6R-mediatedsignalling, and/or of the biological pathways mechanisms, responsesand/or effects in which IL-6, IL-6R and/or IL-6- or IL-6R mediatedsignalling are involved.

As such, the amino acid sequences, polypeptides and compositions of theinvention may for example bind an epitope that lies in, forms part of,or overlaps with (i.e. in the primary or tertiary structure) or is inclose proximity to (i.e. in the primary or tertiary structure) to theIL-6 binding site on IL-6R (for example, competitively with IL-6); bindan epitope that lies in, forms part of, or overlaps with (i.e. in theprimary or tertiary structure) or is in close proximity to (i.e. in theprimary or tertiary structure) the gp130 binding site on IL-6R (forexample, competitively with gp130); and/or bind an epitope that lies in,forms part of, or overlaps with (i.e. in the primary or tertiarystructure) or is in close proximity to (i.e. in the primary or tertiarystructure) the gp 130 binding site on the complex (for example,competitively with gp130). Preferably, any such epitope is anextracellular epitope. Some specific epitopes to which the amino acidsequences, Nanobodies and polypeptides of the invention may preferablybind will become clear from the further description herein.

As such, the amino acid sequences, polypeptides and compositions of thepresent invention can be used for the prevention and treatment ofdiseases and disorders associated with IL-6R, IL-6 and/or with theIL-6/IL-6R complex (optionally in further complex with gp130), and/orwith the signaling pathway(s) and/or the biological functions andresponses in which IL-6 and/or the IL-6/IL-6R complex (optionally infurther complex with gp130) are involved, and in particular for theprevention and treatment of diseases and disorders associated withIL-6R, IL-6 and/or with the IL-6/IL-6R complex (optionally in furthercomplex with gp130), and/or with the signaling pathway(s) and/or thebiological functions and responses in which IL-6R, IL-6 and/or with theIL-6/IL-6R complex (optionally in further complex with gp130) areinvolved, which are characterized by excessive and/or unwantedsignalling mediated by IL-6R or by the pathway(s) in which IL-6R isinvolved. Examples of such diseases and disorders associated with IL-6R,IL-6 and/or with the IL-6/IL-6R complex, and/or with the signalingpathway(s) and/or the biological functions and responses in which IL-6and/or the IL-6/IL-6R complex are involved, will be clear to the skilledperson based on the disclosure herein, and for example include thefollowing diseases and disorders: sepsis (Starnes et al., 1999) andvarious forms of cancer such as multiple myeloma disease (MM), renalcell carcinoma (RCC), plasma cell leukaemia (Klein et al., 1991),lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer.Non-limiting examples of other diseases caused by excessive IL-6production or signalling include bone resorption (osteoporosis) (Roodmanet al., 1992; Jilka et al., 1992), cachexia (Strassman et al., 1992),psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma,AIDS-related lymphoma (Emilie et al., 1994), inflammatory diseases anddisorder such as rheumatoid arthritis, systemic onset juvenileidiopathic arthritis, hypergammaglobulinemia (Grau et al., 1990);Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE),multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma,asthma (in particular allergic asthma) and autoimmune insulin-dependentdiabetes mellitus (Campbell et al., 1991). Other IL-6R, IL-6 and/orIL-6/IL-6R complex related disorders will be clear to the skilledperson. Such diseases and disorders are also generally referred toherein as “IL-6R related disorders”.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can for example be used to prevent and/orto treat all diseases and disorders that are currently being preventedor treated with active principles that can modulate IL-6R-mediatedsignalling, such as those mentioned in the prior art cited above. It isalso envisaged that the polypeptides of the invention can be used toprevent and/or to treat all diseases and disorders for which treatmentwith such active principles is currently being developed, has beenproposed, or will be proposed or developed in future. In addition, it isenvisaged that, because of their favourable properties as furtherdescribed herein, the polypeptides of the present invention may be usedfor the prevention and treatment of other diseases and disorders thanthose for which these known active principles are being used or will beproposed or developed; and/or that the polypeptides of the presentinvention may provide new methods and regimens for treating the diseasesand disorders described herein.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

DETAILED DESCRIPTION OF THE INVENTION

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of one or more IL-6Rrelated disorders (as defined herein); and to provide methods for thediagnosis, prevention and/or treatment of such diseases and disordersthat involve the administration and/or use of such agents andcompositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of one or more IL-6R related disorders (asdefined herein); and to provide methods for the diagnosis, preventionand/or treatment of such diseases and disorders that involve theadministration and/or the use of such therapeutic proteins andcompositions.

Accordingly, it is a specific object of the present invention to provideamino acid sequences and polypeptides that are directed against (asdefined herein) IL-6R, in particular against IL-6R from a warm-bloodedanimal, more in particular against IL-6R from a mammal, and especiallyagainst human IL-6R; and to provide proteins and polypeptides comprisingor essentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat are suitable for prophylactic, therapeutic and/or diagnostic use ina warm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat can be used for the prevention, treatment, alleviation and/ordiagnosis of one or more IL-6R related disorders (as defined herein) ina warm-blooded animal, in particular in a mammal, and more in particularin a human being.

It is also a specific object of the invention to provide such amino acidsequences and such proteins and/or polypeptides that can be used in thepreparation of pharmaceutical or veterinary compositions for theprevention and/or treatment of one or more IL-6R related disorders (asdefined herein) in a warm-blooded animal, in particular in a mammal, andmore in particular in a human being.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, polypeptides and compositions thatare described herein.

In general, the invention provides amino acid sequences and polypeptidesthat are directed against (as defined herein) and/or can specificallybind (as defined herein) to IL-6R; as well as compounds and constructs,and in particular proteins and polypeptides, that comprise at least onesuch amino acid sequence.

More in particular, the invention provides amino acid sequences that canbind to IL-6R with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to IL-6R with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to IL-6R with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁴ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to IL-6R with a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to IL-6R with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to IL-6R will become clear from thefurther description and examples herein.

For binding to IL-6R, an amino acid sequence of the invention willusually contain within its amino acid sequence one or more amino acidresidues or one or more stretches of amino acid residues (i.e. with each“stretch” comprising two or amino acid residues that are adjacent toeach other or in close proximity to each other, i.e. in the primary ortertiary structure of the amino acid sequence) via which the amino acidsequence of the invention can bind to IL-6R, which amino acid residuesor stretches of amino acid residues thus form the “site” for binding toIL-6R (also referred to herein as as the “antigen binding site”).

The amino acid sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more amino acid sequences of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than IL-6R), so as to provide a monovalent,multivalent or multispecific polypeptide of the invention, respectively,all as described herein. Such a protein or polypeptide may also be inessentially isolated form (as defined herein).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the amino acid sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human IL-6R; whereas for veterinary purposes, the amino acidsequences and polypeptides of the invention are preferably directedagainst IL-6R from the species to be treated, or at at leastcross-reactive with IL-6R from the species to be treated.

Furthermore, an amino acid sequence of the invention (or compound,construct or polypeptide comprising one or more such amino acidsequences) may optionally, and in addition to the at least one bindingsite for binding against IL-6R, contain one or more further bindingsites for binding against other antigens, proteins or targets.

The efficacy of the amino acid sequences and polypeptides of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable assays and animal modelswill be clear to the skilled person, and for example includeproliferation assays using IL6-dependent cell lines including B9, XG1and 7TD1, collagen induced arthritis model, transplant model of synovialtissue in SCID mice, xenograft models of various human cancers,including lymphoma, myeloma, prostate cancer and renal cell carcinoma,IBD models including TNBS, DSS and IL10 knockout models, as well as theassays and animal models used in the experimental part below and in theprior art cited herein (Peake et al., Rheumatology 2006; 45(12):1485-9;Wahid et al.; Clin Exp Immunol. 2000, 122:133-142; Matsuno et al.,Arthritis and rheumatism, 1998, 41: 2014-2021).

Also, according to the invention, amino acid sequences and polypeptidesthat are directed against IL-6R from a first species of warm-bloodedanimal may or may not show cross-reactivity with IL-6R from one or moreother species of warm-blooded animal, by which is meant that these aminoacid sequences are also “directed against” (as defined herein) and/orare capable of specific binding to (as defined herein) IL-6R from saidwarm-blooded animal. For example, amino acid sequences and polypeptidesdirected against human IL-6R may or may not show cross reactivity withIL-6R from one or more other species of primates (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus)) and/or with IL-6R from one or morespecies of animals that are often used in animal models for diseases(for example mouse, rat, rabbit, pig or dog), and in particular inanimal models for diseases and disorders associated with IL-6R (such asthe species and animal models mentioned herein). In this respect, itwill be clear to the skilled person that such cross-reactivity, whenpresent, may have advantages from a drug development point of view,since it allows the amino acid sequences and polypeptides against humanIL-6R to be tested in such disease models. In a preferred butnon-limiting aspect, the amino acid sequences of the invention (as wellas compounds, constructs and polypeptides comprising the same) may becross-reactive with the amino acid sequence for IL-6R from Macacafascicularis that is given in SEQ ID NO: 633. For this sequence and thecorresponding cDNA sequence, reference is also made to thenon-prepublished US provisional application filed by Ablynx N. V. onJul. 19, 2007 entitled “Receptor for interleukin-6 (IL-6) from Macacafascicularis”; see SEQ ID NO: 3 and FIG. 1B for the cDNA sequence andSEQ ID NO: 4 and FIG. 3B for the amino acid sequence.

More generally, amino acid sequences and polypeptides of the inventionthat are cross-reactive with IL-6R from multiple species of mammal willusually be advantageous for use in veterinary applications, since itwill allow the same amino acid sequence or polypeptide to be used acrossmultiple species. Thus, it is also encompassed within the scope of theinvention that amino acid sequences and polypeptides directed againstIL-6R from one species of animal (such as amino acid sequences andpolypeptides against human IL-6R) can be used in the treatment ofanother species of animal, as long as the use of the amino acidsequences and/or polypeptides provide the desired effects in the speciesto be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of IL-6Ragainst which the amino acid sequences and polypeptides of the inventionare directed. However, it is generally assumed and preferred that theamino acid sequences and polypeptides of the invention are preferablydirected against any epitope of the IL-6 receptor involved in theinteraction of the IL-6 receptor with IL-6.

Such epitopes or interaction sites have been described in detail inBoulanger et al. (2003) (Science 300, 2101-2104) and reference isspecifically made to FIG. 2 in cited reference. More preferably, theamino acid sequences and polypeptides of the present invention aredirected against an extracellular domain of the IL-6 receptor. Stillmore preferably, the amino acid sequences and polypeptides of thepresent invention are directed against the extracellular D3 domain ofthe IL-6 receptor. Still more preferably, the amino acid sequences andpolypeptides of the present invention interact with one or more of the18 contact residues as described in Boulanger et al. 2003 (Science 300,2101-2104) present in the extracellular D3 domain of the IL-6 receptorthat contribute to the interaction of the IL-6 receptor with IL-6. Mostpreferably, the amino acid sequences and polypeptides of the presentinvention interact with amino acid residues Phe229 and Phe279 present inthe extracellular D3 domain of the IL-6 receptor.

Thus, in one preferred, but non-limiting aspect, the amino acidsequences and polypeptides of the invention are directed against anyepitope of the IL-6 receptor involved in the interaction of the IL-6receptor with IL-6, and are as further defined herein.

Alternatively, the amino acid sequences and polypeptides of theinvention are directed against any epitope of the IL-6 receptor involvedin the interaction of the IL-6 receptor with gp130. Such epitopes orinteraction sites have been described in detail in Boulanger et al.(2003) (Science 300, 2101-2104) and reference is specifically made toFIG. 2 in cited reference.

In this context, according to a non-limiting aspect, amino acidsequences and polypeptides of the invention are preferably such thatthey can compete for binding to the IL-6 receptor with the commerciallyavailable human-mouse reconstituted chimeric monoclonal anti-IL6Rantibody Tocilizumab (MRA) (Chugai/Roche) or an antigen binding fragmentthereof (see for example WO 92/19759 and corresponding European patentEP 0628639, as well as Shinkura et al., 1998, Anticancer Research 18,1217-1222), for example in the assay described in Example 29; and/orsuch that they can bind to the same epitope or binding site on IL-6R asTocilizumab, or to an epitope close to said binding site and/oroverlapping with said binding site.

Also, according to a non-limiting aspect, amino acid sequences andpolypeptides of the invention are preferably such that they can competefor binding to the IL-6 receptor with the reference IgG and/or referenceFab according to patent EP 0628639; and/or such that they can bind tothe same epitope or binding site on IL-6R as said reference IgG orreference Fab, or to an epitope close to said binding site and/oroverlapping with said binding site. For the preparation and sequence ofsaid reference IgG and reference Fab, reference is made to ReferenceExample 1 below, as well as to SEQ ID NO's: 629 to 632.

Thus, generally and without limitation, amino acid sequences andpolypeptides of the invention may be directed against any epitope of theIL-6 receptor involved in the interaction of the IL-6 receptor with IL-6and/or gp130.

The amino acid sequences and polypeptides of the invention are alsopreferably (but without limitation) such that they effect a decrease(i.e. by at least 1 percent such as by at least 10 percent or more) inthe levels of C-reactive protein (CRP) in a mammal (such as a humansubject or in a suitable animal model for inflammation such as thecynomolgus monkey model used in the experimental part below) when theyare administered to said mammal in a therapeutically relevant amount.

It is also within the scope of the invention that, where applicable, anamino acid sequence or polypeptide of the invention can bind to two ormore antigenic determinants, epitopes, parts, domains, subunits orconfirmations of IL-6R. In such a case, the antigenic determinants,epitopes, parts, domains or subunits of IL-6R to which the amino acidsequences and/or polypeptides of the invention bind may be essentiallythe same (for example, if IL-6R contains repeated structural motifs oroccurs in a multimeric form) or may be different (and in the lattercase, the amino acid sequences and polypeptides of the invention maybind to such different antigenic determinants, epitopes, parts, domains,subunits of IL-6R with an affinity and/or specificity which may be thesame or different). Also, for example, when IL-6R exists in an activatedconformation and in an inactive conformation, the amino acid sequencesand polypeptides of the invention may bind to either one of theseconfirmation, or may bind to both these confirmations (i.e. with anaffinity and/or specificity which may be the same or different). Also,for example, the amino acid sequences and polypeptides of the inventionmay bind to a conformation of IL-6R in which it is bound to a pertinentligand, may bind to a conformation of IL-6R in which it not bound to apertinent ligand, or may bind to both such conformations (again with anaffinity and/or specificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofIL-6R; or at least to those analogs, variants, mutants, alleles, partsand fragments of IL-6R that contain one or more antigenic determinantsor epitopes that are essentially the same as the antigenicdeterminant(s) or epitope(s) to which the amino acid sequences andpolypeptides of the invention bind in IL-6R (e.g. in wild-type IL-6R).Again, in such a case, the amino acid sequences and polypeptides of theinvention may bind to such analogs, variants, mutants, alleles, partsand fragments with an affinity and/or specificity that are the same as,or that are different from (i.e. higher than or lower than), theaffinity and specificity with which the amino acid sequences of theinvention bind to (wild-type) IL-6R. It is also included within thescope of the invention that the amino acid sequences and polypeptides ofthe invention bind to some analogs, variants, mutants, alleles, partsand fragments of IL-6R, but not to others.

When IL-6R exists in a monomeric form and in one or more multimericforms, it is within the scope of the invention that the amino acidsequences and polypeptides of the invention only bind to IL-6R inmonomeric form, only bind to IL-6R in multimeric form, or bind to boththe monomeric and the multimeric form. Again, in such a case, the aminoacid sequences and polypeptides of the invention may bind to themonomeric form with an affinity and/or specificity that are the same as,or that are different from (i.e. higher than or lower than), theaffinity and specificity with which the amino acid sequences of theinvention bind to the multimeric form.

Also, when IL-6R can associate with other proteins or polypeptides toform protein complexes (e.g. with multiple subunits), it is within thescope of the invention that the amino acid sequences and polypeptides ofthe invention bind to IL-6R in its non-associated state, bind to IL-6Rin its associated state, or bind to both. In all these cases, the aminoacid sequences and polypeptides of the invention may bind to suchmultimers or associated protein complexes with an affinity and/orspecificity that may be the same as or different from (i.e. higher thanor lower than) the affinity and/or specificity with which the amino acidsequences and polypeptides of the invention bind to IL-6R in itsmonomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more amino acid sequences directed against IL-6R maybind with higher avidity to IL-6R than the corresponding monomeric aminoacid sequence(s). For example, and without limitation, proteins orpolypeptides that contain two or more amino acid sequences directedagainst different epitopes of IL-6R may (and usually will) bind withhigher avidity than each of the different monomers, and proteins orpolypeptides that contain two or more amino acid sequences directedagainst IL-6R may (and usually will) bind also with higher avidity to amultimer of IL-6R.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of IL-6R (including monomeric, multimericand associated forms) that are the most relevant from a biologicaland/or therapeutic point of view, as will be clear to the skilledperson.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against IL-6R; and more preferably willbe capable of specific binding to IL-6R, and even more preferablycapable of binding to IL-6R with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein.Some non-limiting examples of such parts, fragments, analogs, mutants,variants, alleles, derivatives, proteins and/or polypeptides will becomeclear from the further description herein. Additional fragments orpolypeptides of the invention may also be provided by suitably combining(i.e. by linking or genetic fusion) one or more (smaller) parts orfragments as described herein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to IL-6R; and more preferably capable of binding to IL-6R withan affinity (suitably measured and/or expressed as a K_(D)-value (actualor apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Also, parts, fragments, analogs,mutants, variants, alleles and/or derivatives of such amino acidsequences are preferably such that they comprise an immunoglobulin foldor are capable for forming, under suitable conditions, an immunoglobulinfold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the amino acid sequences of theinvention may be naturally occurring amino acid sequences (from anysuitable species) or synthetic or semi-synthetic amino acid sequences.In a specific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable speies) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized V_(HH) sequences or Nanobodies), “camelized” (as definedherein) immunoglobulin sequences, as well as immunoglobulin sequencesthat have been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody® (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 October 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence of the invention may be aNanobody® (as defined herein) or a suitable fragment thereof. [Note:Nanobody®, Nanobodies® and Nanoclone® are registered trademarks ofAblynx N. V.] Such Nanobodies directed against IL-6R will also bereferred to herein as “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to thefurther description below, as well as to the prior art cited herein. Inthis respect, it should however be noted that this description and theprior art mainly described Nanobodies of the so-called “V_(H)3 class”(i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which Nanobodies form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of Nanobody directed against IL-6R, and for example alsocovers the Nanobodies belonging to the so-called “V_(H)4 class” (i.e.Nanobodies with a high degree of sequence homology to human germlinesequences of the V_(H)4 class such as DP-78), as for example describedin the U.S. provisional application 60/792,279 by Ablynx N. V. entitled“DP-78-like Nanobodies” filed on Apr. 14, 2006.

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which one or more of the Hallmark residuesare as further defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which the framework sequences are as furtherdefined herein.

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against IL-6R, to suitable fragmentsthereof, as well as to polypeptides that comprise or essentially consistof one or more of such Nanobodies and/or suitable fragments.

SEQ ID NO's 399 to 471 give the amino acid sequences of a number ofV_(HH) sequences that have been raised against IL-6R.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to IL-6R and which:

-   a) have 80% amino acid identity with at least one of the amino acid    sequences of SEQ ID NO's: 399 to 471, in which for the purposes of    determining the degree of amino acid identity, the amino acid    residues that form the CDR sequences are disregarded. In this    respect, reference is also made to Table A-1, which lists the    framework 1 sequences (SEQ ID NO's: 42 to 92), framework 2 sequences    (SEQ ID NO's: 144 to 194), framework 3 sequences (SEQ ID NO's: 246    to 296) and framework 4 sequences (SEQ ID NO's: 348 to 398) of the    Nanobodies of SEQ ID NO's: 399 to 471 (with respect to the amino    acid residues at positions 1 to 4 and 27 to 30 of the framework 1    sequences, reference is also made to the comments made below. Thus,    for determining the degree of amino acid identity, these residues    are preferably disregarded);    and in which:-   b) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below (it being understood that V_(HH) sequences will contain    one or more Hallmark residues; and that partially humanized    Nanobodies will usually, and preferably, [still] contain one or more    Hallmark residues [although it is also within the scope of the    invention to provide—where suitable in accordance with the    invention—partially humanized Nanobodies in which all Hallmark    residues, but not one or more of the other amino acid residues, have    been humanized]; and that in fully humanized Nanobodies, where    suitable in accordance with the invention, all amino acid residues    at the positions of the Hallmark residues will be amino acid    residues that occur in a human V_(H)3 sequence. As will be clear to    the skilled person based on the disclosure herein that such V_(HH)    sequences, such partially humanized Nanobodies with at least one    Hallmark residue, such partially humanized Nanobodies without    Hallmark residues and such fully humanized Nanobodies all form    aspects of this invention).

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be amino acid sequences that areas generally defined for Nanobodies in the previous paragraphs, but inwhich at least one amino acid residue is present (and in particular, inat least one of the framework residues) that is and/or that correspondsto a humanizing substitution (as defined herein). Some preferred, butnon-limiting humanizing substitutions (and suitable combinationsthereof) will become clear to the skilled person based on the disclosureherein. In addition, or alternatively, other potentially usefulhumanizing substitutions can be ascertained by comparing the sequence ofthe framework regions of a naturally occurring V_(HH) sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said V_(HH) sequence (in any manner known per se, asfurther described herein) and the resulting humanized V_(HH) sequencescan be tested for affinity for the target, for stability, for ease andlevel of expression, and/or for other desired properties. In this way,by means of a limited degree of trial and error, other suitablehumanizing substitutions (or suitable combinations thereof) can bedetermined by the skilled person based on the disclosure herein. Also,based on the foregoing, (the framework regions of) a Nanobody may bepartially humanized or fully humanized.

Some particularly preferred humanized Nanobodies of the invention arehumanized variants of the Nanobodies of SEQ ID NO's: 399 to 471.

Thus, some other preferred Nanobodies of the invention are Nanobodieswhich can bind (as further defined herein) to IL-6R and which:

-   a) are a humanized variant of one of the amino acid sequences of SEQ    ID NO's: 399 to 471; and/or-   b) have 80% amino acid identity with at least one of the amino acid    sequences of SEQ ID NO's: 399 to 471, in which for the purposes of    determining the degree of amino acid identity, the amino acid    residues that form the CDR sequences are disregarded;    and in which:-   c) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below

Some preferred, but non-limiting examples of humanized Nanobodies of theInvention are given in SEQ ID NO's: 609 to 628.

According to another specific aspect of the invention, the inventionprovides a number of streches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to IL-6R. Thesestreches of amino acid residues may be present in, and/or may becorporated into, an amino acid sequence of the invention, in particularin such a way that they form (part of) the antigen binding site of anamino acid sequence of the invention. As these streches of amino acidresidues were first generated as CDR sequences of heavy chain antibodiesor V_(HH) sequences that were raised against IL-6R (or may be based onand/or derived from such CDR sequences, as further described herein),they will also generally be referred to herein as “CDR sequences” (i.e.as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively). Itshould however be noted that the invention in its broadest sense is notlimited to a specific structural role or function that these streches ofamino acid residues may have in an amino acid sequence of the invention,as long as these stretches of amino acid residues allow the amino acidsequence of the invention to bind to IL-6R. Thus, generally, theinvention in its broadest sense comprises any amino acid sequence thatis capable of binding to IL-6R and that comprises one or more CDRsequences as described herein and, and in particular a suitablecombination of two or more such CDR sequences, that are suitably linkedto each other via one or more further amino acid sequences, such thatthe entire amino acid sequence forms a binding domain and/or bindingunit that is capable of binding to IL-6R. It should however also benoted that the presence of only one such CDR sequence in an amino acidsequence of the invention may by itself already be sufficient to providean amino acid sequence of the invention that is capable of binding toIL-6R; reference is for example again made to the so-called “Expeditefragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences thatare described herein (or any suitable combination thereof). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1 sequences, CDR2 sequencesand CDR3 sequences that are described herein (or any suitablecombination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531). Alternatively, such anamino acid sequence may be a suitable “protein scaffold” that comprisesleast one stretch of amino acid residues that corresponds to such a CDRsequence (i.e. as part of its antigen binding site). Suitable scaffoldsfor presenting amino acid sequences will be clear to the skilled person,and for example comprise, without limitation, to binding scaffolds basedon or derived from immunoglobulins (i.e. other than the immunoglobulinsequences already described herein), protein scaffolds derived fromprotein A domains (such as Affibodies™), tendamistat, fibronectin,lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimersand PDZ domains (Binz et al, Nat. Biotech 2005, Vol 23:1257), andbinding moieties based on DNA or RNA including but not limited to DNA orRNA aptamers (Ulrich et al._Comb Chem High Throughput Screen 20069(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to IL-6R, and more in particular such that itcan bind to IL-6R with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect ofthe invention may be any amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast two amino acid sequences that are chosen from the group consistingof the CDR1 sequences described herein, the CDR2 sequences describedherein and the CDR3 sequences described herein, such that (i) when thefirst amino acid sequence is chosen from the CDR1 sequences describedherein, the second amino acid sequence is chosen from the CDR2 sequencesdescribed herein or the CDR3 sequences described herein; (ii) when thefirst amino acid sequence is chosen from the CDR2 sequences describedherein, the second amino acid sequence is chosen from the CDR1 sequencesdescribed herein or the CDR3 sequences described herein; or (iii) whenthe first amino acid sequence is chosen from the CDR3 sequencesdescribed herein, the second amino acid sequence is chosen from the CDR1sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention maybe amino acid sequences that comprise at least one antigen binding site,wherein said antigen binding site comprises at least three amino acidsequences that are chosen from the group consisting of the CDR1sequences described herein, the CDR2 sequences described herein and theCDR3 sequences described herein, such that the first amino acid sequenceis chosen from the CDR1 sequences described herein, the second aminoacid sequence is chosen from the CDR2 sequences described herein, andthe third amino acid sequence is chosen from the CDR3 sequencesdescribed herein. Preferred combinations of CDR1, CDR2 and CDR3sequences will become clear from the further description herein. As willbe clear to the skilled person, such an amino acid sequence ispreferably an immunoglobulin sequence (as further described herein), butit may for example also be any other amino acid sequence that comprisesa suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against IL-6R, that comprises one ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 93 to 143;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;-   d) the amino acid sequences of SEQ ID NO's: 195 to 245;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: SEQ ID    NO's: 195 to 245;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;-   g) the amino acid sequences of SEQ ID NO's: 297 to 347;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 93 to 143;-   ii) the amino acid sequences of SEQ ID NO's: 195 to 245; and-   iii) the amino acid sequences of SEQ ID NO's: 297 to 347;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against IL-6R.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against IL-6R, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 93 to 143;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;-   d) the amino acid sequences of SEQ ID NO's: 195 to 245;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;-   g) the amino acid sequences of SEQ ID NO's: 297 to 347;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 93 to 143;-   ii) the amino acid sequences of SEQ ID NO's: 195 to 245; and-   iii) the amino acid sequences of SEQ ID NO's: 297 to 347;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 93 to    143, the second stretch of amino acid residues corresponds to one of    the amino acid sequences of SEQ ID NO's: 195 to 245 or of SEQ ID    NO's: 297 to 347; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 195    to 245, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 93 to 143 or of SEQ ID    NO's: 297 to 347; or (iii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 297 to 347, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 93 to    143 or of SEQ ID NO's: 195 to 245.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against IL-6R.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against IL-6R, that comprises threeor more stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 93 to 143;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 195 to 245;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 297 to 347;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 93 to 143; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 195 to 245; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:297 to 347.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against IL-6R.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ ID NO's399 to 471

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to IL-6R; and more in particularbind to IL-6R with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 93 to 143;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;    and/or

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 195 to 245;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;    and/or

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 297 to 347;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 93 to 143; and/or CDR2 is chosen from thegroup consisting of the amino acid sequences of SEQ ID NO's: 195 to 245;and/or CDR3 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 297 to 347.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 93 to 143;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 93 to    143;    and

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 195 to 245;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 195 to    245;    and

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 297 to 347;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 297 to    347; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 93 to 143; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 195 to 245; andCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 297 to 347.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to IL-6R; and more in particularbind to IL-6R with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 399 to 471. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid residue and one or more of the sequences of SEQ ID NO's: 399 to471, in which the amino acid residues that form the framework regionsare disregarded. Such amino acid sequences of the invention can be asfurther described herein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanzed) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a Nanobody® (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a Nanobody®. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

Again, as generally described herein for the amino acid sequences of theinvention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions). For a further description ofthese “Expedite fragments”, reference is again made to WO 03/050531).

TABLE A-1 Preferred combinations of CDR and framework sequences. CLONEID FR1 ID CDR1 ID FR2 ID CDR2 PMP40C9(t) 42 EVQLVESGGGLVQPG 93 YYAIG 144WFRQAPGK 195 CMDSSAGTT GSLRLSCAASGFSLD EREGVS STYYSDSVKG PMP34F8(t) 43EVQLVESGGGLVQPG 94 YYAIG 145 WFRQAPGK 196 CMDSSDGTT GSLRLSCAASGFSLDEREGVS NTYYSDSVKG PMP34E9(t) 44 EVQLVESGGGLVQPG 95 YYAIG 146 WFRQAPGK197 CISSSDGSTY GSLRLSCAASGFTLD EREGVS YADSVKG PMP34D2(t) 45EVQLVESGGGLVQPG 96 YFAIG 147 WFRQAPGK 198 CISSSDGSTY GSLRLSCAASGFTLDERERVS YADSVKG PMP34C3(t) 46 EVQLVESGGGLVQPG 97 YYVIG 148 WFRQAPGK 199CISSSDGSTY GSLRLSCVASGFSLD EREGVS YADSVKG PMP34A5(t) 47 EVQLVESGGGLVQPG98 YFAIG 149 WFRQAPGK 200 CISSSDGSTY GSLRLSCAASGFTLG EREGVS YADSVKGPMP33G3(t) 48 EVQLVESGGGLVQ 99 YFAIG 150 WFRQAPG 201 CISSSDGSAPGGSLRLSCAASG KEREGVS YYADSVKG FTLG PMP33C10(t) 49 EVQLVESGGGLVQ 100DYGMS 151 WVRQAPG 202 AISWNGGS PGGSLRLSCAASG KGLEWVS TYYTESMKG FTFDPMP33A2(t) 50 EVQLVESGGGLVQPG 101 YYAIG 152 WFRQAPG 203 CMDSSGGTTGSLRLSCAASGFSLD KEREGVS STYYSDSVKG PMP32H5(t) 51 EVQLVESGGGLVQPG 102SYDMS 153 WVRQAPG 204 AINSGGGST GSLRLSCAASGFTFG KGPEWVS YYADSVKGPMP32F10(t) 52 EVQLVESGGGLVQAG 103 DYAIG 154 WFRQAPG 205 CISSSDGSTGSLRLSCAASGFTFD KEREGIS YYADSVKG PMP31F4(t) 53 EVQLVESGGGLVQPG 104 SYDMS155 WVRQAPG 206 AINSGGGST GSLRLSCAASGFTFG KGPEWVS YYADSVKG PMP31D2(t) 54EVQLVESGGGLVQAG 105 DYAIG 156 WFRQAPG 207 GISSSDGNT GSLRLSCAASGFTFDKEREGVS YYADSVKG PMP31C8(t) 55 EVQLVESGGGLVQPG 106 SYDMS 157 WVRQAPG 208AINSGGGST GSLRLSCAASGFTFG KGPEWVS YYADSVKG PMP31C5(t) 56 EVQLVESGGGLVQAG107 DYAIG 158 WFRQAPG 209 CISSSDGST GSLRLSCAASGFTFD KEREGVS YYADSVKGPMP31B4(t) 57 EVQLVESGGGLVQPG 108 SYDMS 159 WVRQAPG 210 AINSGGGSTGSLRLSCAASGFTFG KGPEWVS YYADSVKG PMP31B11(t) 58 EVQLVESGGGLVQPG 109SYDMS 160 WVRQAPG 211 AINSGGGST GSLRLSCAASGFTFG KGPEWVS YYADSVKGPMP30G11(t) 59 EVQLVESGGGLVQPG 110 YYVIG 161 WFRQAPG 212 CISSSDGSTYGSLRLSCVASGFSLD KEREGVS YADSVKG PMP30B6(t) 60 EVQLVESGGGLVQPG 111 YYVIG162 WFRQAPG 213 CISSSDRSTY GSLRLSCAASGFTLD KEREAVA YADSVKG PMP30B1(t) 61EVQLVESGGGLVQPG 112 YYAIG 163 WWRQAPG 214 CISSGDGST GSLRLSCAASGFTLDKGREGVS NYADSVKG PMP30A2(t) 62 EVQLVESGGGLVQPG 113 YYVIG 164 WFRQAPG 215CIGSSDDST GSLRLSCAASGFTLD KEREGVS YYADSVKG PMP30A10(t) 63EVQLVESGGGLVQPG 114 DYGMS 165 WVRQAPG 216 AISWNGGST GSLRLSCAASGFTFDKGLEWVS YYTESMKG PMP28H6(t) 64 EVQLVESGGGLVQPG 115 SYDMS 166 WVRQAPG 217AINSGGGST GSLRLSCAASGFTFG KGPEWVS YYADSVKG PMP28F7(t) 65 EVQLVESGGGLVQPG116 SYDMS 167 WVRQAPG 218 AINSGGGST GSLRLSCAASGFTFG KGPEWVS YYADSVKGPMP28D4(t) 66 EVQLVESGGGLVQPG 117 SYDMS 168 WVRQAPG 219 AINSGGGSTGSLRLSCAASGFTFG KGPEWVS YYADSVKG PMP28C7(t) 67 EVQLVESGGGLVQPG 118 SYDMS169 WVRQAPG 220 AINSGGDNTY GSLRLSCAASGFTFG KGPEWVS YADSVKG PMP28B1(t) 68EVQLVESGGGLVQPG 119 YYAIG 170 WFRQAPG 221 CISSSDGSTY GSLRLSCAASGFTLNKEREGVS YADSVKG PMP28A2(t) 69 EVQLVESGGGLVQPG 120 SYDMS 171 WVRQAPG 222AINSGGGSTY GSLRLSCAASGFTFG KGPEWVS YADSVKG PMP40H5 70 EVQLVESGGGLVQPG121 YYAIG 172 WFRQAPG 223 CMDSSSGTT GSLRLSCAASGFSLD KEREGVS STYYSDSVKGPMP35H4 71 EVQLVESGGGLVQPG 122 DYGMS 173 WVRQAPG 224 AISWNGNNTYGSLRLSCAASGFTFD RATEWVS YTESMKG PMP35F4 72 EVQLVESGGGLVQAG 123 SYDMG 174WYRQAPG 225 IITWNSSTYYA GSLRLSCAASGRTFS KEREFVA DSVKG PMP35E11 73EVQLVESGGGLVQAG 124 DYAIG 175 WFRQAPG 226 CISSSDGSTY GSLRLSCAASGFTFDKEHEGVS YADSVKG PMP35C10 74 EVQLVESGGGLVQAG 125 SYDMG 176 WYRQAPG 227VIHWSSGSTY GSLRLSCAASGRTFS KEREFVA YADPVKG PMP34G9 75 EVQLVESGGGLVQAG126 SYDMT 177 WYRQVPG 228 VISWSGGSTY GSLRLSCAASGRTSS KEREFVA YADSVKGPMP34G3 76 EVQLVESGGGLVQPG 127 YYAIG 178 WFRQAPG 229 CISSSDGSTYGSLRLSCAASGFTLD KERERVS YADSVKG PMP34E10 77 EVQLVESGGGLVQAG 128 SYAMG179 WGRQAPG 230 TISWSGGST GSLRLSCAASGRTFS KEREFVA YYADSVKG PMP34C11 78EVQLVESGGGLVQPG 129 YSAIG 180 WFRQAPG 231 CISGSDGST GSLRLSCAAAGFTLDKEREMFS WYADSVAG PMP34A12 79 EVQLVESGGGLVQPG 130 YYVIG 181 WFRQAPG 232CISSSDGSTY GSLRLSCVASGFSLD KEREGVS YADSVKG PMP33A3 80 EVQLVESGGGLVQPG131 YGAIG 182 WFRQAPG 233 CISSSTGSTY GSLRLSCAASGFTLD KEREGVS YADSVKGPMP32E2 81 EVQLVESGGGLVQAG 132 DNTMGWT 183 WNRQPPG 234 IIATDGSTNYGSLRLSCAASGNIFD KQRELVA ADSVKG PMP32E10 82 EVQLVESGGGLVQPG 133 SYDMS 184WVRQAPG 235 AINSGGGST GSLRLSCAASGFTFG KGPEWVS YYADSVKG PMP32C9 83EVQLVESGGGLVQAG 134 DYDIG 185 WFRQAPG 236 GISSSDGNT GSLRLSCAASGFTFDKEREGVS YYADSVKG PMP31A4 84 EVQLVESGGGLVQAG 135 VNAMG 186 WYRQAPG 237GIISGGSTNY GSLRLSCAASGSIFK KQRELVA ADSVKG PMP30C11 85 EVQLVESGGGLVQAG136 SYDMG 187 WYRQAPG 238 VISRSGSSTY GSLRLSCAASGRTFS KEREFVA YADSVKGPMP28G3 86 EVQLVESGGGLVQAG 137 TETMG 188 WYRQPPG 239 ATITHGGTTNGSLRLSCTASGNIFS KQRDVV YADSVKG PMP28E11 87 EVQLVESGGGFVQAG 138 INRMG 189WYRQALG 240 IITNHGSTNY GSLRLSCIASGDNFS KQRELVA ADAVKG 059B.IL6R.cl5.7(t)88 EVQLVESGGGLVQAG 139 GADAG 190 WNRQTPG 241 AINWSGNST GSLRLSCAASGRTFSKEREFVA YYADSVKG 059A.IL6Rcl4(t) 89 EVQLVESGGGLVQAG 140 SYDMG 191WYRQGPG 242 AISWSGGGT GSLRLSCAASGRTLS KEREFVA DYVDSVKG 059A.IL6Rcl3(t)90 EVQLVESGGGLVQPG 141 SYAIG 192 WFRQAPG 243 CISTSDGSTY GSLRLSCAASGFTLDKEPEGVS YADSVKG 059A.IL6Rcl2(t) 91 EVQLVESGGGLVQAG 142 NIAMG 193WIREAPGK 244 ALTWSGGST GSLRLSCAASGRTFS EREFVA YYADSVKG 059A.IL6Rcl1(t)92 EVQLVESGGGLVQAG 143 DFAIG 194 WFRQAPG 245 CISSSDGSTY GSLRLSCAASGLTDDKEPEGVS YADSVKG CLONE ID FR3 ID CDR3 ID FR4 PMP40C9(t) 246 RFTISRDDAKN297 DGHLNWGQ 348 WGQGTQ TVYLQMNSLKP RYVPCSQIS VTVSS EDTAVYYCAA WRGWNDYPMP34F8(t) 247 RFTISRDDAKN 298 DGHLNWGQ 349 WGQGTQ TVYLQMNSLKP PYVPCSQISVTVSS EDTASYYCAA WRGWNDY PMP34E9(t) 248 RFTISRDNAKN 299 DRSVYYCSG 350WGQGTQ TVYLQMNSLKP DAPEEYY VTVSS EDTAAYYCAT PMP34D2(t) 249 RFTISRDNAKN300 DRSVYYCSG 351 WGQGTQ TVYLQMNSLKP GAPEEYY VTVSS EDTAVYYCAT PMP34C3(t)250 RFTISRDNAKN 301 DLLRTPEFC 352 WGRGTQ TVYLQMNSLKP VDSAPYDY VTVSSEDTAVYYCAA PMP34A5(t) 251 RFTVSRDNAK 302 DRSVYYCSG 353 WGQGTQNTVYLQMNSLK GAPEEYY VTVSS PEDTAVYYCAT PMP33G3(t) 252 RFTVSRDNA 303DRSVYYCS 354 WGQGT KNTVYLQMN GGAPEEYY QVTVSS SLKPEDTAVY YCAT PMP33C10(t)253 RFTISRDNAK 304 GSTAIVGV 355 WGQGT NTVYLQMNS PPTYPDEY QVTVSSLKPEDTAVYY DY CVK PMP33A2(t) 254 RFTISRDDAKN 305 DGHLNWGQ 356 WGQGTTVYLQMNSLKP RYVPCSQIS QVTVSS EDTAVYYCAA WRGWNDY PMP32H5(t) 255RFTISRDNAKN 306 DWRYSDYDL 357 WGQGT TLYLQMNSLKP PLPPPGDY QVTVSSEDTAVYYCAT PMP32F10(t) 256 RFTISSDNAKN 307 EPPDSSWIL 358 WGQGTTVYLQMNSLKP DGSPEFFKF QVTVSS EDTAVYYCAA PMP31F4(t) 257 RFTISRDNAKN 308DWRYSDYDL 359 WGQGT TLYLQMNSLKP PLPPPGDY QVTVSS EDTAVYYCAT PMP31D2(t)258 RFTISSDNAKN 309 EPPDSNWYL 360 WGQGT TVYLQMNSLKP DGSPEFFKF QVTVSSEDTAVYYCAA PMP31C8(t) 259 RFTISRDNAKN 310 DWRYSDYDL 361 WGQGTTLYLQMNSLKP PLPPPGDY QVTVSS EDTAVYYCAT PMP31C5(t) 260 RFTISSDNAKN 311EPPDSMWSL 362 WGQGT TVYLLMNSLKP DGSPEFFKF QVTVSS EDTAVYYCAA PMP31B4(t)261 RFTISRDNAKN 312 DWRYSDYDL 363 WGQGT TLYLQMNSLKP PLPPPGDY QVTVSSEDTAVYYCAT PMP31B11(t) 262 RFTISRDNAKN 313 DWRYSDYDL 364 WGQGTQTLYLQMNSLKP PLPPPGDY VTVSS EDTAVYYCAT PMP30G11(t) 263 RFTISRDNAKN 314DLLRTPEFC 365 WGQGTQ TVYLQMNSLKP VDSAPYDY VTVSS EDTAVYYCAA PMP30B6(t)264 RFTISRDNAKN 315 DLLRTPEFC 366 WGQGTQ TGYLQMNSLK SDSAPYDY VTVSSPEDTAVYYCAA PMP30B1(t) 265 RFTISRDNAKN 316 DRSVYYCSG 367 WGQGTQTVYLQMNSLKP GAPEEYY VTVSS EDTAVYYCAT PMP30A2(t) 266 RFTISRDNAKN 317DLLRTPEFCT 368 WGQGTQ TVYLQMNSLKP DSAPYDY VTVSS EDTAVYYCAA PMP30A10(t)267 RFTISRDNAKN 318 GSTAIVGVPP 369 WGQGTQ TVYLQMNSLKP TYPDEYDY VTVSSEDTAVYYCVK PMP28H6(t) 268 RFTISRDNAKN 319 DWRYSDYDL 370 WGQGTQTLYLQMNSLKP PLPPPGDY VTVSS EDTAVYYCAT PMP28F7(t) 269 RFTISRDNAKN 320DWRYSDYDL 371 WGQGTQ TLYLQMNSLKP PLPPPGDY VTVSS EDTAVYYCAT PMP28D4(t)270 RFTISRDNAKN 321 DWRYSDYDL 372 WGQGTQ TLYLQMNSLKP PLPPPGDY VTVSSEDTAVYYCAT PMP28C7(t) 271 RFTISRDNAKNT 322 DWRYSDYDLP 373 WGQGTQLYLQMNSLKPED LPPPGDY VTVSS TAVYYCAT PMP28B1(t) 272 RFTISRDNAKNT 323EGLGDSDSPC 374 WGQGTQ FYLQMNSLKPED GAAWYNDY VTVSS TAVYYCAA PMP28A2(t)273 RFTISRDNAKNT 324 DWRYSDYDLP 375 WGQGTQ LYLQMNSLKPED LPPPGDY VTVSSTAVYYCAT PMP40H5 274 RFTISRDDAKNT 325 DGHLNWGQR 376 WGQGTQ VYLQMNSLKPEDYVPCSQISWR VTVSS TAVYYCAA GWNDY PMP35H4 275 RFTISRDNAKNT 326 GSTAIVGVPPT377 WGQGTQ VYLQMNSLKPED YPDEYDY VTVSS TAVYYCVK PMP35F4 276 RFTISRDNAKNT327 QYGLGYAEDY 378 WGQGTQ VYLQMNSLKPED VTVSS TAIYYCNA PMP35E11 277RFTISSDNAKNT 328 ERDVPARSLC 379 RGQGTQ VYLQMNSLKPED GSYYWYDY VTVSSTAVYYCAA PMP35C10 278 RFTISRDNAKNT 329 FLPGPEGFHDY 380 WGQGTQVYLQMNSLKPED VTVSS TAIYYCNA PMP34G9 279 RFTISRDNAKNT 330 YTGGGDDY 381WGQGTQ VYLQMNSLKPED VTVSS TAIYYCNA PMP34G3 280 RFTISRDNAKN 331 DRSVYYCSG382 WGQGTQ TVYLQMNSLKP GAPEEYY VTVSS EDTAAYYCAT PMP34E10 281 RFTISRDNAKN332 DLAEFKYSD 383 WGQGTQ TVYLQMNSLKP YADY VTVSS EDTAVYYCAA PMP34C11 282RFTISFDNAKN 333 TGGVRGPCA 384 WGQGTQ TVYLQMNSLKP YEYEY VTVSS EDTGLYICAVPMP34A12 283 RFTISRDNAKN 334 DLLRTPEFC 385 WGQGTQ TVYLQMNSLKP VDSAPYDYVTVSS EDTAVYYCAA PMP33A3 284 RFTISRDNGKN 335 DKMWSPCL 386 WGQGTQTVYLQMNSLKP VAANEEALF VTVSS EDTAVYYCAA EYDY PMP32E2 285 RFTISRDNAKN 336FSLRLGRDY 387 WGQGTQ TVYLQMNSLKP VTVSS EDTAVYYCNL PMP32E10 286RFTISRDNAKN 337 DWRYSDYDL 388 WGQGTQ TLYLQMNSLKP PLPPPGDY VTVSSEDTAVYYCAT PMP32C9 287 RFTISSDNAKN 338 EPPDSSWYL 389 WGQGTQ TVYLQMNSLKPDGSPEFFKY VTVSS EDTAVYYCAA PMP31A4 288 RLTISRDNAKN 339 VTTNSDYDL 390WGQGTQ TVYLQMNSLKP GRDY VTVSS EDTAVYYCSF PMP30C11 289 RFTISRDNAKNT 340EVVAGD 391 WGQGTQV VYLQMNSLKPED YDY TVSS TAIYYCKA PMP28G3 290RFTISRDNRKNT 341 RSSWYS 392 WGQGTQV VYLQMNSLKPED PEY TVSS TGVYYCNAPMP28E11 291 RFTISRDYAKNTV 342 YISEVGT 393 WGQGIQV YLQMNGLKPDDT WRDDYTVSS AVYYCNA 059B.IL6R.cl5.7(t) 292 RFTVSRDNAKNT 343 FRDDYYS 394EGKGTLVT VYLQMNSLKPED VSS TAVYYCHA 059A.IL6Rcl4(t) 293 RFTISRDTAKNT 344LGTTDSD 395 WGQGTQV MYLQMNSLKPED YEGELY TVSS TAIYYCNA 059A.IL6Rcl3(t)294 RFTISRDNAKNT 345 DGGPHA 396 WGQGTQV VYLQMNSLKPED PLTVQD TVSSTAVYYCTA MCVMAIA DY 059A.IL6Rcl2(t) 295 RFTISRDSAKNTV 346 DEEIHLIV 397WGQGTQV YLQMNKLKPEDT SISIADF TVSS AVYYCVA 059A.IL6Rcl1(t) 296RFTISSDNAKNTV 347 LFDRCGS 398 WGKGTLV YLQMNSLKPEDT TWYYGM TVSS AVYFCTADY

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moreamino acid sequences of the invention (or suitable fragments thereof),and optionally further comprises one or more other groups, residues,moieties or binding units. As will become clear to the skilled personfrom the further disclosure herein, such further groups, residues,moieties, binding units or amino acid sequences may or may not providefurther functionality to the amino acid sequence of the invention(and/or to the compound or construct in which it is present) and may ormay not modify the properties of the amino acid sequence of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb”'s, amino acid sequences that aresuitable for use as a dAb, or Nanobodies®.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences of the invention so as to provide a “derivative” ofan amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivates as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise amino acid sequences or polypeptides ofthe invention that have been chemically modified to increase thehalf-life thereof (for example, by means of pegylation); amino acidsequences of the invention that comprise at least one additional bindingsite for binding to a serum protein (such as serum albumin); orpolypeptides of the invention that comprise at least one amino acidsequence of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the amino acid sequence of the invention. Examples ofpolypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more amino acid sequencesof the invention are suitable linked to one or more serum proteins orfragments thereof (such as (human) serum albumin or suitable fragmentsthereof) or to one or more binding units that can bind to serum proteins(such as, for example, domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies can bind to serum proteins such as serum albumin (such ashuman serum albumin), serum immunoglobulins such as IgG, ortransferrine; reference is made to the further description andreferences mentioned herein); polypeptides in which an amino acidsequence of the invention is linked to an Fc portion (such as a humanFc) or a suitable part or fragment thereof; or polypeptides in which theone or more amino acid sequences of the invention are suitable linked toone or more small proteins or peptides that can bind to serum proteins(such as, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489).

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hour, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and mayfor example be in the form of a genetic construct, as further describedherein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention (or asuitable fragment thereof), at least one polypeptide of the inventionand/or at least one nucleic acid of the invention, and optionally one ormore further components of such compositions known per se, i.e.depending on the intended use of the composition. Such a product orcomposition may for example be a pharmaceutical composition (asdescribed herein), a veterinary composition or a product or compositionfor diagnostic use (as also described herein). Some preferred butnon-limiting examples of such products or compositions will become clearfrom the further description herein.

In another specific, but non-limiting aspect, the amino acid sequencesand polypeptides described herein are such that they (a) specificallybind (as defined herein) to the IL-6 receptor; and (b) are capable ofdownregulating the IL-6 receptor and/or are capable of inhibiting,decreasing or downregulating the signalling of the IL-6 receptor and/orthe pathway(s), mechanism(s) or signalling in which the IL-6 or IL-6R isinvolved. As will be clear to the skilled person, such an amino acidsequence or polypeptide can generally be used as an antagonist of IL-6,of the IL-6 receptor and/or of the biological pathways, mechanisms oreffects in which IL-6, Il-6R and/or Il-6/IL-6R mediated signalling isinvolved. Any such decrease or downregulation (which can be at least 1%,such as at least 5%, or more than 10%, or up to 50% or 100% or more in arelevant parameter, compared to the same parameter under conditions inwhich the amino acid sequence or polypeptide is not bound to the IL-6receptor), may be measured in any suitable manner known per se, forexample using one of the assays used in the Experimental Part and/ormentioned herein.

For example, such antagonistic amino acid sequences and polypeptides maybe competitive of non-competitive inhibitors of the binding of IL-6 toIL-6R.

More in particular, and in addition to (a) and (b) above, and optionallyin addition to (d) and/or (e) below, such antagonistic amino acidsequences and polypeptides may bind to IL-6R in such a way that (c)binding of IL-6 to IL-6R is blocked, inhibited or reduced; compared tothe binding of IL-6 to its receptor without the presence of the aminoacid sequence or Nanobody of the invention.

For example, and without limitation, such antagonistic amino acidsequences and polypeptides may bind to or close to the IL-6 om IL-6R.

Also, in addition to (a) and (b) above, and optionally in addition to(c) above or (e) below, such antagonistic amino acid sequences andpolypeptides may bind to IL-6R (i.e. as such or as present in theIL-6/IL-6R complex) in such a way that (d) the formation of theIL-6/IL-6R complex is inhibited or affected (e.g. fully or partiallydisrupted) in such a way that the binding of the complex to—e.g. itsaffinity for—gp130 is reduced (or reversely, that the binding of gp 130to—e.g. its affinity for—the complex is reduced), so that the signalinginduced/mediated by the binding of the complex to gp130 is modulated(e.g. reduced); compared to the formation of the complex and its bindingto gp130 without the presence of the amino acid sequence or Nanobody ofthe invention.

Also, in addition to (a) and (b) above, and optionally in addition to(c) or (d) above, such antagonistic amino acid sequences andpolypeptides may bind to IL-6R (i.e. as such or as present in theIL-6/IL-6R complex) in such a way that (e) binding to IL-6R (i.e. assuch or as present in the IL-6/IL-6R complex) in such a way that theformation of the IL-6/IL-6R complex essentially is not affected but thatthe binding of said complex to gp130 is modulated (e.g. inhibited), sothat the signalling induced/mediated by the binding of the complex togp130 is modulated (e.g. reduced); compared to the formation of thecomplex and its binding to gp130 without the presence of the amino acidsequence or Nanobody of the invention.

Alternatively, such antagonistic amino acid sequences and polypeptidesmay bind to another epitope, site, domain or region on the IL-6 receptor(e.g. allosteric binding) such that the IL-6 receptor becomes lesssensitive for binding of IL-6 (and/or that the signalling of the IL-6receptor upon binding of IL-6 is reduced).

It is also possible that such antagonistic amino acid sequences andpolypeptides may bind to another epitope, site, domain or region on theIL-6 receptor such that the ligand-mediated dimerization of the growthfactor receptor is prevented, reduced or inhibited.

Accordingly, in the context of the present invention, “modulating” or“to modulate” generally means exercising an agonistic or antagonisticeffect, respectively, with respect to IL-6, IL-6R and/or the biologicalpathways, responses, signalling, mechanisms or effects in which IL-6and/or IL-6R is involved. In particular, “modulating” or “to modulate”may mean either an such an agonistic or antagonistic effect (i.e. a fullor partial agonistic or antagonistic effect, respectively), as measuredusing a suitable in vitro, cellular or in vivo assay (such as thosementioned herein), that leads to a change in a relevant parameter by atleast 1%, preferably at least 5%, such as at least 10% or at least 25%,for example by at least 50%, at least 60%, at least 70%, at least 80%,or 90% or more, compared to same parameter in the same assay under thesame conditions but without the presence of the amino acid sequence,Nanobody or polypeptide of the invention.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of amino acid sequences    for amino acid sequences that can bind to and/or have affinity for    IL-6R;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for IL-6R.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with IL-6R or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for IL-6R;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized withIL-6R or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820.

In another aspect, the method for generating an amino acid sequencedirected against IL-6R may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for IL-6R;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with IL-6R or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) nucleotidesequences encoding amino acid sequences will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises the oneof the above methods and in addition at least the steps of determiningthe nucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally viaone ormore suitable linkers) so as to provide a polypeptide of the invention.Also, a nucleic acid sequence encoding an amino acid sequence of theinvention may be suitably humanized (or alternatively camelized) andsuitably expressed; and/or one or more nucleic acid sequences encodingan amino acid sequence of the invention may be linked to each other orto one or more nucleic acid sequences that encode other suitable aminoacid sequences (optionally via nucleotide sequences that encode one ormore suitable linkers), after which the nucleotide sequence thusobtained may be suitably expressed so as to provide a polypeptide of theinvention.

The invention further relates to applications and uses of the amino acidsequences, polypeptides, nucleic acids, host cells, products andcompositions described herein, as well as to methods for the preventionand/or treatment for diseases and disorders associated with IL-6R. Somepreferred but non-limiting applications and uses will become clear fromthe further description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the Nanobodies of the invention and of polypeptides of the inventioncomprising the same, which form some of the preferred aspects of theinvention.

As will become clear from the further description herein, Nanobodiesgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the Nanobodies of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The above and other aspects, embodiments and advantages of the inventionwill become clear from the further description hereinbelow, in which:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.),    Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et    al, eds., “Current protocols in molecular biology”, Green Publishing    and Wiley Interscience, New York (1987); Lewin, “Genes II”, John    Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of    Gene Manipulation: An Introduction to Genetic Engineering”, 2nd    edition, University of California Press, Berkeley, Calif. (1981);    Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh    (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.    Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology”    (6th Ed.), Garland Science Publishing/Churchill Livingstone, N.Y.    (2005), as well as to the general background art cited herein;-   b) Unless indicated otherwise, the term “immunoglobulin    sequence”—whether used herein to refer to a heavy chain antibody or    to a conventional 4-chain antibody—is used as a general term to    include both the full-size antibody, the individual chains thereof,    as well as all parts, domains or fragments thereof (including but    not limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H)/V_(L) domains, respectively). In addition, the term    “sequence” as used herein (for example in terms like “immunoglobulin    sequence”, “antibody sequence”, “variable domain sequence”, “V_(HH)    sequence” or “protein sequence”), should generally be understood to    include both the relevant amino acid sequence as well as nucleic    acid sequences or nucleotide sequences encoding the same, unless the    context requires a more limited interpretation;-   c) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein;-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table    A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar AlanineAla A uncharge Valine Val V (at pH Leucine Leu L 6.0-7.0)⁽³⁾ IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH ThreonineThr T 6.0-7.0) Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH Histidine⁽⁴⁾ HisH 6.0-7.0) Aspartate Asp D Glutamate Glu E Notes: a) Sometimes alsoconsidered to be a polar uncharged amino acid. b) Sometimes alsoconsidered to be a nonpolar uncharged amino acid. c) As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person. d)As is known in the art, the charge of a His residue is greatly dependantupon even small shifts in pH, but a His residu can generally beconsidered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated by    dividing [the number of nucleotides in the first nucleotide sequence    that are identical to the nucleotides at the corresponding positions    in the second nucleotide sequence] by [the total number of    nucleotides in the first nucleotide sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of a nucleotide in the second nucleotide sequence—compared to the    first nucleotide sequence—is considered as a difference at a single    nucleotide (position).-    Alternatively, the degree of sequence identity between two or more    nucleotide sequences may be calculated using a known computer    algorithm for sequence alignment such as NCBI Blast v2.0, using    standard settings.-    Some other techniques, computer algorithms and settings for    determining the degree of sequence identity are for example    described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318,    WO 00/78972, WO 98/49185 and GB 2 357 768-A.-    Usually, for the purpose of determining the percentage of “sequence    identity” between two nucleotide sequences in accordance with the    calculation method outlined hereinabove, the nucleotide sequence    with the greatest number of nucleotides will be taken as the “first”    nucleotide sequence, and the other nucleotide sequence will be taken    as the “second” nucleotide sequence;-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (as refered to herein as    “amino acid identity”) may be calculated by dividing [the number of    amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.-    Alternatively, the degree of sequence identity between two amino    acid sequences may be calculated using a known computer algorithm,    such as those mentioned above for determining the degree of sequence    identity for nucleotide sequences, again using standard settings.-    Usually, for the purpose of determining the percentage of “sequence    identity” between two amino acid sequences in accordance with the    calculation method outlined hereinabove, the amino acid sequence    with the greatest number of amino acid residues will be taken as the    “first” amino acid sequence, and the other amino acid sequence will    be taken as the “second” amino acid sequence.-    Also, in determining the degree of sequence identity between two    amino acid sequences, the skilled person may take into account    so-called “conservative” amino acid substitutions, which can    generally be described as amino acid substitutions in which an amino    acid residue is replaced with another amino acid residue of similar    chemical structure and which has little or essentially no influence    on the function, activity or other biological properties of the    polypeptide. Such conservative amino acid substitutions are well    known in the art, for example from WO 04/037999, GB-A-3 357 768, WO    98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or    combinations of such substitutions may be selected on the basis of    the pertinent teachings from WO 04/037999 as well as WO 98/49185 and    from the further references cited therein.-    Such conservative substitutions preferably are substitutions in    which one amino acid within the following groups (a)-(e) is    substituted by another amino acid residue within the same group: (a)    small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,    Pro and Gly; (b) polar, negatively charged residues and their    (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively    charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar    residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues:    Phe, Tyr and Trp.-    Particularly preferred conservative substitutions are as follows:    Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His;    Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala    or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu    into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into    Leu, into Tyr or into Be; Phe into Met, into Leu or into Tyr; Ser    into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into    Val, into Be or into Leu.-    Any amino acid substitutions applied to the polypeptides described    herein may also be based on the analysis of the frequencies of amino    acid variations between homologous proteins of different species    developed by Schulz et al., Principles of Protein Structure,    Springer-Verlag, 1978, on the analyses of structure forming    potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974    and Adv. Enzymol, 47: 45-149, 1978, and on the analysis of    hydrophobicity patterns in proteins developed by Eisenberg et al.,    Proc. Nad. Acad Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J    Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev.    Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their    entirety by reference. Information on the primary, secondary and    tertiary structure of Nanobodies is given in the description herein    and in the general background art cited above. Also, for this    purpose, the crystal structure of a V_(HH) domain from a llama is    for example given by Desmyter et al., Nature Structural Biology,    Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology    (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361    (1999). Further information about some of the amino acid residues    that in conventional V_(H) domains form the V_(H)/V_(L) interface    and potential camelizing substitutions on these positions can be    found in the prior art cited above.-   g) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length;-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences;-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this may mean that the latter    nucleotide sequence or amino acid sequence has been incorporated    into the firstmentioned nucleotide sequence or amino acid sequence,    respectively, but more usually this generally means that the    firstmentioned nucleotide sequence or amino acid sequence comprises    within its sequence a stretch of nucleotides or amino acid residues,    respectively, that has the same nucleotide sequence or amino acid    sequence, respectively, as the latter sequence, irrespective of how    the firstmentioned sequence has actually been generated or obtained    (which may for example be by any suitable method described herein).    By means of a non-limiting example, when a Nanobody of the invention    is said to comprise a CDR sequence, this may mean that said CDR    sequence has been incorporated into the Nanobody of the invention,    but more usually this generally means that the Nanobody of the    invention contains within its sequence a stretch of amino acid    residues with the same amino acid sequence as said CDR sequence,    irrespective of how said Nanobody of the invention has been    generated or obtained. It should also be noted that when the latter    amino acid sequence has a specific biological or structural    function, it preferably has essentially the same, a similar or an    equivalent biological or structural function in the firstmentioned    amino acid sequence (in other words, the firstmentioned amino acid    sequence is preferably such that the latter sequence is capable of    performing essentially the same, a similar or an equivalent    biological or structural function). For example, when a Nanobody of    the invention is said to comprise a CDR sequence or framework    sequence, respectively, the CDR sequence and framework are    preferably capable, in said Nanobody, of functioning as a CDR    sequence or framework sequence, respectively. Also, when a    nucleotide sequence is said to comprise another nucleotide sequence,    the firstmentioned nucleotide sequence is preferably such that, when    it is expressed into an expression product (e.g. a polypeptide), the    amino acid sequence encoded by the latter nucleotide sequence forms    part of said expression product (in other words, that the latter    nucleotide sequence is in the same reading frame as the    firstmentioned, larger nucleotide sequence).-   j) A nucleic acid sequence or amino acid sequence is considered to    be “(in) essentially isolated (form)”—for example, compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid sequence or amino acid    sequence is considered “essentially isolated” when it has been    purified at least 2-fold, in particular at least 10-fold, more in    particular at least 100-fold, and up to 1000-fold or more. A nucleic    acid sequence or amino acid sequence that is “in essentially    isolated form” is preferably essentially homogeneous, as determined    using a suitable technique, such as a suitable chromatographical    technique, such as polyacrylamide-gel electrophoresis;-   k) The term “domain” as used herein generally refers to a globular    region of an antibody chain, and in particular to a globular region    of a heavy chain antibody, or to a polypeptide that essentially    consists of such a globular region. Usually, such a domain will    comprise peptide loops (for example 3 or 4 peptide loops)    stabilized, for example, as a sheet or by disulfide bonds. 1) The    term ‘antigenic determinant’ refers to the epitope on the antigen    recognized by the antigen-binding molecule (such as a Nanobody or a    polypeptide of the invention) and more in particular by the    antigen-binding site of said molecule. The terms “antigenic    determinant” and “epitope” may also be used interchangeably herein.-   m) An amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein. In particular, an amino    acid sequence that “against” or “directed against” an antigenic    determinant, epitope, antigen or protein (or for at least one part,    fragment or epitope thereof) and/or that can specifically bind to,    that has affinity for and/or that has specificity for a specific    antigenic determinant, epitope, antigen or protein (or for at least    one part, fragment or epitope thereof) is defined herein as an amino    acid sequence that can bind to said antigenic determinant, epitope,    antigen or protein (or for at least one part, fragment or epitope    thereof) with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²    moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or    less and more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an    association constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and    preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸    to 10¹² liter/moles).-   n) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as a    Nanobody or a polypeptide of the invention) molecule can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity. The affinity, represented by the    equilibrium constant for the dissociation of an antigen with an    antigen-binding protein (K_(D)), is a measure for the binding    strength between an antigenic determinant and an antigen-binding    site on the antigen-binding protein: the lesser the value of the    K_(D), the stronger the binding strength between an antigenic    determinant and the antigen-binding molecule (alternatively, the    affinity can also be expressed as the affinity constant (K_(A)),    which is 1/K_(D)). As will be clear to the skilled person (for    example on the basis of the further disclosure herein), affinity can    be determined in a manner known per se, depending on the specific    antigen of interest. Avidity is the measure of the strength of    binding between an antigen-binding molecule (such as a Nanobody or    polypeptide of the invention) and the pertinent antigen. Avidity is    related to both the affinity between an antigenic determinant and    its antigen binding site on the antigen-binding molecule and the    number of pertinent binding sites present on the antigen-binding    molecule. Typically, antigen-binding proteins (such as the amino    acid sequences, Nanobodies and/or polypeptides of the invention)    will bind to their antigen with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter    (i.e. with an association constant (K_(A)) of 10⁵ to 10¹²    liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more    and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin sequence of the invention    will bind to the desired serum protein with an affinity less than    500 nM, preferably less than 200 nM, more preferably less than 10    nM, such as less than 500 pM. Specific binding of an antigen-binding    protein to an antigen or antigenic determinant can be determined in    any suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as    radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein.-    The dissociation constant may be the actual or apparent    dissociation constant, as will be clear to the skilled person.    Methods for determining the dissociation constant will be clear to    the skilled person, and for example include the techniques mentioned    herein. In this respect, it will also be clear that it may not be    possible to measure dissociation constants of more then 10⁻⁴    moles/liter or 10⁻³ moles/liter (e,g, of 10⁻² moles/liter).    Optionally, as will also be clear to the skilled person, the (actual    or apparent) dissociation constant may be calculated on the basis of    the (actual or apparent) association constant (K_(A)), by means of    the relationship [K_(D)=1/K_(A)].-    The affinity denotes the strength or stability of a molecular    interaction. The affinity is commonly given as by the K_(D), or    dissociation constant, which has units of mol/liter (or M). The    affinity can also be expressed as an association constant, K_(A),    which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or M⁻¹). In the    present specification, the stability of the interaction between two    molecules (such as an amino acid sequence, Nanobody or polypeptide    of the invention and its intended target) will mainly be expressed    in terms of the K_(D) value of their interaction; it being clear to    the skilled person that in view of the relation K_(A)=1/K_(D),    specifying the strength of molecular interaction by its K_(D) value    can also be used to calculate the corresponding K_(A) value. The    K_(D)-value characterizes the strength of a molecular interaction    also in a thermodynamic sense as it is related to the free energy    (DG) of binding by the well known relation DG=RT. ln(K_(D))    (equivalently DG=−RT. ln(K_(A))), where R equals the gas constant, T    equals the absolute temperature and In denotes the natural    logarithm.-    The K_(D) for biological interactions which are considered    meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M (0.1    nM) to 10⁻⁵M (10000 nM). The stronger an interaction is, the lower    is its K_(D).-    The K_(D) can also be expressed as the ratio of the dissociation    rate constant of a complex, denoted as k_(off), to the rate of its    association, denoted k_(on) (so that K_(D)=k_(off)/k_(on) and    K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s    is the SI unit notation of second). The on-rate k_(on) has units    M⁻¹s⁻¹. The on-rate may vary between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹    s⁻¹, approaching the diffusion-limited association rate constant for    bimolecular interactions. The off-rate is related to the half-life    of a given molecular interaction by the relation    t_(1/2)=1n(2)/k_(off). The off-rate may vary between 10⁻⁶ s−1 (near    irreversible complex with a t_(1/2) of multiple days) to 1s⁻¹    (t_(1/2)=0.69 s).-    The affinity of a molecular interaction between two molecules can    be measured via different techniques known per se, such as the well    the known surface plasmon resonance (SPR) biosensor technique (see    for example Ober et al., Intern. Immunology, 13, 1551-1559, 2001)    where one molecule is immobilized on the biosensor chip and the    other molecule is passed over the immobilized molecule under flow    conditions yielding k_(on), k_(off) measurements and hence K_(D) (or    K_(A)) values. This can for example be performed using the    well-known BIACORE instruments.-    It will also be clear to the skilled person that the measured K_(D)    may correspond to the apparent K_(D) if the measuring process    somehow influences the intrinsic binding affinity of the implied    molecules for example by artifacts related to the coating on the    biosensor of one molecule. Also, an apparent K_(D) may be measured    if one molecule contains more than one recognition sites for the    other molecule. In such situation the measured affinity may be    affected by the avidity of the interaction by the two molecules.-    Another approach that may be used to assess affinity is the 2-step    ELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et    al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes    a solution phase binding equilibrium measurement and avoids possible    artifacts relating to adsorption of one of the molecules on a    support such as plastic.-    However, the accurate measurement of K_(D) may be quite    labor-intensive and as consequence, often apparent K_(D) values are    determined to assess the binding strength of two molecules. It    should be noted that as long all measurements are made in a    consistent way (e.g. keeping the assay conditions unchanged)    apparent K_(D) measurements can be used as an approximation of the    true K_(D) and hence in the present document K_(D) and apparent    K_(D) should be treated with equal importance or relevance. Finally,    it should be noted that in many situations the experienced scientist    may judge it to be convenient to determine the binding affinity    relative to some reference molecule. For example, to assess the    binding strength between molecules A and B, one may e.g. use a    reference molecule C that is known to bind to B and that is suitably    labeled with a fluorophore or chromophore group or other chemical    moiety, such as biotin for easy detection in an ELISA or FACS    (Fluorescent activated cell sorting) or other format (the    fluorophore for fluorescence detection, the chromophore for light    absorption detection, the biotin for streptavidin-mediated ELISA    detection). Typically, the reference molecule C is kept at a fixed    concentration and the concentration of A is varied for a given    concentration or amount of B. As a result an IC₅₀ value is obtained    corresponding to the concentration of A at which the signal measured    for C in absence of A is halved. Provided K_(D ref), the K_(D) of    the reference molecule, is known, as well as the total concentration    c_(ref) of the reference molecule, the apparent K_(D) for the    interaction A-B can be obtained from following formula:    K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if cref<<K_(D) ref,    K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in a    consistent way (e.g. keeping c_(ref) fixed) for the binders that are    compared, the strength or stability of a molecular interaction can    be assessed by the IC₅₀ and this measurement is judged as equivalent    to K_(D) or to apparent K_(D) throughout this text.-   o) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as the time taken for the    serum concentration of the amino acid sequence, compound or    polypeptide to be reduced by 50%, in vivo, for example due to    degradation of the sequence or compound and/or clearance or    sequestration of the sequence or compound by natural mechanisms. The    in vivo half-life of an amino acid sequence, compound or polypeptide    of the invention can be determined in any manner known per se, such    as by pharmacokinetic analysis. Suitable techniques will be clear to    the person skilled in the art, and may for example generally involve    the steps of suitably administering to a warm-blooded animal (i.e.    to a human or to another suitable mammal, such as a mouse, rabbit,    rat, pig, dog or a primate, for example monkeys from the genus    Macaca (such as, and in particular, cynomologus monkeys (Macaca    fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon    (Papio ursinus)) a suitable dose of the amino acid sequence,    compound or polypeptide of the invention; collecting blood samples    or other samples from said animal; determining the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention in said blood sample; and calculating, from (a plot    of) the data thus obtained, the time until the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention has been reduced by 50% compared to the initial level    upon dosing. Reference is for example made to the Experimental Part    below, as well as to the standard handbooks, such as Kenneth, A et    al: Chemical Stability of Pharmaceuticals: A Handbook for    Pharmacists and Peters et al, Pharmacokinete analysis: A Practical    Approach (1996). Reference is also made to “Pharmacokinetics”, M    Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition    (1982).-    As will also be clear to the skilled person (see for example pages    6 and 7 of WO 04/003019 and in the further references cited    therein), the half-life can be expressed using parameters such as    the t1/2-alpha, t1/2-beta and the area under the curve (AUC).-    In the present specification, an “increase in half-life” refers to    an increase in any one of these parameters, such as any two of these    parameters, or essentially all three these parameters. As used    herein “increase in half-life” or “increased half-life” in    particular refers to an increase in the t1/2-beta, either with or    without an increase in the t1/2-alpha and/or the AUC or both.-   p) As also further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a Nanobody are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   q) The amino acid residues of a Nanobody are numbered according to    the general numbering for V_(H) domains given by Kabat et al.    (“Sequence of proteins of immunological interest”, US Public Health    Services, NIH Bethesda, Md., Publication No. 91), as applied to    V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2):185-195    (see for example FIG. 2 of said article or referred to herein.    According to this numbering, FR1 of a Nanobody comprises the amino    acid residues at positions 1-30, CDR1 of a Nanobody comprises the    amino acid residues at positions 31-35, FR2 of a Nanobody comprises    the amino acids at positions 36-49, CDR2 of a Nanobody comprises the    amino acid residues at positions 50-65, FR3 of a Nanobody comprises    the amino acid residues at positions 66-94, CDR3 of a Nanobody    comprises the amino acid residues at positions 95-102, and FR4 of a    Nanobody comprises the amino acid residues at positions 103-113. [In    this respect, it should be noted that—as is well known in the art    for V_(H) domains and for V_(HH) domains—the total number of amino    acid residues in each of the CDR's may vary and may not correspond    to the total number of amino acid residues indicated by the Kabat    numbering (that is, one or more positions according to the Kabat    numbering may not be occupied in the actual sequence, or the actual    sequence may contain more amino acid residues than the number    allowed for by the Kabat numbering). This means that, generally, the    numbering according to Kabat may or may not correspond to the actual    numbering of the amino acid residues in the actual sequence.    Generally, however, it can be said that, according to the numbering    of Kabat and irrespective of the number of amino acid residues in    the CDR's, position 1 according to the Kabat numbering corresponds    to the start of FR1 and vice versa, position 36 according to the    Kabat numbering corresponds to the start of FR2 and vice versa,    position 66 according to the Kabat numbering corresponds to the    start of FR3 and vice versa, and position 103 according to the Kabat    numbering corresponds to the start of FR4 and vice versa.].-    Alternative methods for numbering the amino acid residues of V_(H)    domains, which methods can also be applied in an analogous manner to    V_(HH) domains from Camelids and to Nanobodies, are the method    described by Chothia et al. (Nature 342, 877-883 (1989)), the    so-called “AbM definition” and the so-called “contact definition”.    However, in the present description, claims and figures, the    numbering according to Kabat as applied to V_(HH) domains by    Riechmann and Muyldermans will be followed, unless indicated    otherwise; and-   r) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74(2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N. V. and Ablynx N. V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N. V. and thefurther published patent applications by Ablynx N. V. Reference is alsomade to the further prior art mentioned in these applications, and inparticular to the list of references mentioned on pages 41-43 of theInternational application WO 06/040153, which list and references areincorporated herein by reference.

In accordance with the terminology used in the above references, thevariable domains present in naturally occurring heavy chain antibodieswill also be referred to as “V_(HH) domains”, in order to distinguishthem from the heavy chain variable domains that are present inconventional 4-chain antibodies (which will be referred to hereinbelowas “V_(H) domains”) and from the light chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNanobodies as single antigen-binding proteins or as antigen-bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spacial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   V_(HH) domains and Nanobodies can be expressed from a single        gene and require no post-translational folding or modifications;    -   V_(HH) domains and Nanobodies can easily be engineered into        multivalent and multispecific formats (as further discussed        herein);    -   V_(HH) domains and Nanobodies are highly soluble and do not have        a tendency to aggregate (as with the mouse-derived        antigen-binding domains described by Ward et al., Nature, Vol.        341, 1989, p. 544);    -   V_(HH) domains and Nanobodies are highly stable to heat, pH,        proteases and other denaturing agents or conditions (see for        example Ewert et al, supra);    -   V_(HH) domains and Nanobodies are easy and relatively cheap to        prepare, even on a scale required for production. For example,        V_(HH) domains, Nanobodies and proteins/polypeptides containing        the same can be produced using microbial fermentation (e.g. as        further described below) and do not require the use of mammalian        expression systems, as with for example conventional antibody        fragments;    -   V_(HH) domains and Nanobodies are relatively small        (approximately 15 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumors and other dense tissues) than such conventional 4-chain        antibodies and antigen-binding fragments thereof;    -   V_(HH) domains and Nanobodies can show so-called cavity-binding        properties (inter alia due to their extended CDR3 loop, compared        to conventional V_(H) domains) and can therefore also access        targets and epitopes not accessable to conventional 4-chain        antibodies and antigen-binding fragments thereof. For example,        it has been shown that V_(HH) domains and Nanobodies can inhibit        enzymes (see for example WO 97/49805; Transue et al., (1998),        supra; Lauwereys et al., (1998), supra.

As mentioned above, the invention generally relates to Nanobodiesdirected against the IL-6 receptor, as well as to polypeptidescomprising or essentially consisting of one or more of such Nanobodies,that can be used for the prophylactic, therapeutic and/or diagnosticpurposes described herein.

As also further described herein, the invention further relates tonucleic acids encoding such Nanobodies and polypeptides, to methods forpreparing such Nanobodies and polypeptides, to host cells expressing orcapable of expressing such Nanobodies or polypeptides, to compositionscomprising such Nanobodies, polypeptides, nucleic acids or host cells,and to uses of such Nanobodies, polypeptides, nucleic acids, host cellsor compositions.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained: (1) by isolating the V_(HH) domain of a naturally occurringheavy chain antibody; (2) by expression of a nucleotide sequenceencoding a naturally occurring V_(HH) domain; (3) by “humanization” (asdescribed herein) of a naturally occurring V_(HH) domain or byexpression of a nucleic acid encoding a such humanized V_(HH) domain;(4) by “camelization” (as described herein) of a naturally occurringV_(H) domain from any animal species, and in particular a from speciesof mammal, such as from a human being, or by expression of a nucleicacid encoding such a camelized V_(H) domain; (5) by “camelisation” of a“domain antibody” or “Dab” as described by Ward et al (supra), or byexpression of a nucleic acid encoding such a camelized V_(H) domain; (6)by using synthetic or semi-synthetic techniques for preparing proteins,polypeptides or other amino acid sequences known per se; (7) bypreparing a nucleic acid encoding a Nanobody using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing. Suitable methods and techniques for performing theforegoing will be clear to the skilled person based on the disclosureherein and for example include the methods and techniques described inmore detail herein.

One preferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against the IL-6receptor. As further described herein, such V_(HH) sequences cangenerally be generated or obtained by suitably immunizing a species ofCamelid with the IL-6 receptor (i.e. so as to raise an immune responseand/or heavy chain antibodies directed against the IL-6 receptor), byobtaining a suitable biological sample from said Camelid (such as ablood sample, serum sample or sample of B-cells), and by generatingV_(HH) sequences directed against the IL-6 receptor, starting from saidsample, using any suitable technique known per se. Such techniques willbe clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains against the IL-6receptor, can be obtained from naïve libraries of Camelid V_(HH)sequences, for example by screening such a library using the IL-6receptor, or at least one part, fragment, antigenic determinant orepitope thereof using one or more screening techniques known per se.Such libraries and techniques are for example described in WO 99/37681,WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improvedsynthetic or semi-synthetic libraries derived from naïve V_(HH)libraries may be used, such as V_(HH) libraries obtained from naïveV_(HH) libraries by techniques such as random mutagenesis and/or CDRshuffling, as for example described in WO 00/43507.

Yet another technique for obtaining V_(HH) sequences directed againstthe IL-6 receptor, involves suitably immunizing a transgenic mammal thatis capable of expressing heavy chain antibodies (i.e. so as to raise animmune response and/or heavy chain antibodies directed against the IL-6receptor), obtaining a suitable biological sample from said transgenicmammal (such as a blood sample, serum sample or sample of B-cells), andthen generating V_(HH) sequences directed against The IL-6 receptor,starting from said sample, using any suitable technique known per se.For example, for this purpose, the heavy chain antibody-expressing miceand the further methods and techniques described in WO 02/085945, WO04/049794, WO 06/008548 and Janssens et al., Proc. Natl. Acad. Sci. USA.2006 Oct. 10; 103(41):15130-5 can be used.

A particularly preferred class of Nanobodies of the invention comprisesNanobodies with an amino acid sequence that corresponds to the aminoacid sequence of a naturally occurring V_(HH) domain, but that has been“humanized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of said naturally occurring V_(HH) sequence (and inparticular in the framework sequences) by one or more of the amino acidresidues that occur at the corresponding position(s) in a V_(H) domainfrom a conventional 4-chain antibody from a human being (e.g. indicatedabove). This can be performed in a manner known per se, which will beclear to the skilled person, for example on the basis of the furtherdescription herein and the prior art on humanization referred to herein.Again, it should be noted that such humanized Nanobodies of theinvention can be obtained in any suitable manner known per se (i.e. asindicated under points (1)-(8) above) and thus are not strictly limitedto polypeptides that have been obtained using a polypeptide thatcomprises a naturally occurring V_(HH) domain as a starting material.

Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain, but that hasbeen “camelized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description herein. Such “camelizing” substitutions arepreferably inserted at amino acid positions that form and/or are presentat the V_(H)-V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see for example WO 94/04678 and Davies andRiechmann (1994 and 1996), supra). Preferably, the V_(H) sequence thatis used as a starting material or starting point for generating ordesigning the camelized Nanobody is preferably a V_(H) sequence from amammal, more preferably the V_(H) sequence of a human being, such as aV_(H)3 sequence. However, it should be noted that such camelizedNanobodies of the invention can be obtained in any suitable manner knownper se (i.e. as indicated under points (1)-(8) above) and thus are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(H) domain as astarting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” Nanobody of the invention,respectively. This nucleic acid can then be expressed in a manner knownper se, so as to provide the desired Nanobody of the invention.Alternatively, based on the amino acid sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, the amino acid sequence ofthe desired humanized or camelized Nanobody of the invention,respectively, can be designed and then synthesized de novo usingtechniques for peptide synthesis known per se. Also, based on the aminoacid sequence or nucleotide sequence of a naturally occurring V_(HH)domain or V_(H) domain, respectively, a nucleotide sequence encoding thedesired humanized or camelized Nanobody of the invention, respectively,can be designed and then synthesized de novo using techniques fornucleic acid synthesis known per se, after which the nucleic acid thusobtained can be expressed in a manner known per se, so as to provide thedesired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example comprise combiningone or more parts of one or more naturally occurring V_(H) sequences(such as one or more FR sequences and/or CDR sequences), one or moreparts of one or more naturally occurring V_(HH) sequences (such as oneor more FR sequences or CDR sequences), and/or one or more synthetic orsemi-synthetic sequences, in a suitable manner, so as to provide aNanobody of the invention or a nucleotide sequence or nucleic acidencoding the same.

Optionally, a Nanobody of the invention may also, and in addition to theat least one binding site for binding against IL-6R, contain one or morefurther binding sites for binding against other antigens, proteins ortargets. For methods and positions for introducing such second bindingsites, teference is for example made to Keck and Huston, BiophysicalJournal, 71, October 1996, 2002-2011; EP 0 640 130; WO 06/07260 and theUS provisional application by Ablynx N. V. entitled “Immunoglobulindomains with multiple binding sites” filed on Nov. 27, 2006.

According to one preferred, but non-limiting aspect of the invention, aNanobody in its broadest sense can be generally defined as a polypeptidecomprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 45    according to the Kabat numbering is a charged amino acid (as defined    herein) or a cysteine residue, and position 44 is preferably an E;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:-   b) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid or a cysteine and the amino acid    residue at position 44 according to the Kabat numbering is    preferably E;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In particular, a Nanobody in its broadest sense can be generally definedas a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody ofthe invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In particular, a Nanobody against the IL-6 receptor, according to theinvention may have the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    a and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a Nanobody can generally be defined as a polypeptidecomprising an amino acid sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of Lor R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q; or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q;    and in which:-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R;    and in which:-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to (a-1) to(a-5) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich;

-   a) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    or in which:-   b) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    and in which:-   b) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q;    and in which:-   b) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified on the basis of thefollowing three groups:

-   a) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW at    positions 44-47 according to the Kabat numbering and Q at position    108 according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a V at position 37, and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. The GLEW group also comprises some GLEW-like sequences    such as those mentioned in Table A-3 below;-   b) The “KERE-group”: Nanobodies with the amino acid sequence KERE or    KQRE (or another KERE-like sequence) at positions 43-46 according to    the Kabat numbering and Q or L at position 108 according to the    Kabat numbering. As further described herein, Nanobodies within this    group usually have a F at position 37, an L or F at position 47; and    can have a W, P, R or S at position 103, and preferably have a W at    position 103;-   c) The “103 P, R, S-group”: Nanobodies with a P, R or S at    position 103. These

Nanobodies can have either the amino acid sequence GLEW at positions44-47 according to the Kabat numbering or the amino acid sequence KEREor KQRE at positions 43-46 according to the Kabat numbering, the lattermost preferably in combination with an F at position 37 and an L or an Fat position 47 (as defined for the KERE-group); and can have Q or L atposition 108 according to the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. havecharacteristics of) two or more of these classes. For example, onespecifically preferred group of Nanobodies has GLEW or a GLEW-likesequence at positions 44-47; P, R or S (and in particular R) at position103; and Q at position 108 (which may be humanized to 108L).

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred embodimentsherein, and are more preferably as defined according to one of the morepreferred embodiments herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred embodimentsherein, and are more preferably as defined according to one of the morepreferred embodiments herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferredembodiments herein, and are more preferably as defined according to oneof the more preferred embodiments herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103P, R, Sresidues mentioned above, the Nanobodies of the invention can contain,at one or more positions that in a conventional V_(H) domain would form(part of) the V_(H)/V_(L) interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2). Such substitutions include, butare not limited to, the GLEW-like sequences mentioned in Table A-3below; as well as the substitutions that are described in theInternational Application WO 00/29004 for so-called “microbodies”, e.g.so as to obtain a Nanobody with Q at position 108 in combination withKLEW at positions 44-47. Other possible substitutions at these positionswill be clear to the skilled person based upon the disclosure herein.

In one embodiment of the Nanobodies of the invention, the amino acidresidue at position 83 is chosen from the group consisting of L, M, S, Vand W; and is preferably L.

Also, in one embodiment of the Nanobodies of the invention, the aminoacid residue at position 83 is chosen from the group consisting of R, K,N, E, G, I, T and Q; and is most preferably either K or E (forNanobodies corresponding to naturally occurring V_(HH) domains) or R(for “humanized” Nanobodies, as described herein). The amino acidresidue at position 84 is chosen from the group consisting of P, A, R,S, D T, and V in one embodiment, and is most preferably P (forNanobodies corresponding to naturally occurring V_(HH) domains) or R(for “humanized” Nanobodies, as described herein).

Furthermore, in one embodiment of the Nanobodies of the invention, theamino acid residue at position 104 is chosen from the group consistingof G and D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table A-3.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(HH) domains arementioned in Table A-4. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE A-3 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; predominantly L L, M, S, V, W; preferably L  37 V, I,F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G G⁽²⁾,E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾or E⁽³⁾  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; preferablyW⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R  83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Qor T; preferably K or R; most preferably K  84 A, T, D; P⁽⁵⁾, A, L, R,S, T, D, V; preferably P predominantly A 103 W W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S;preferably W 104 G G or D; preferably G 108 L, M or T; Q, L⁽⁷⁾ or R;preferably Q or L⁽⁷⁾ predominantly L Notes: a) In particular, but notexclusively, in combination with KERE or KQRE at positions 43-46. b)Usually as GLEW at positions 44-47. c) Usually as KERE or KQRE atpositions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG atpositions 43-47. Alternatively, also sequences such as TERE (for exampleTEREL), KECE (for example KECEL or KECER), RERE (for example REREG),QERE (for example QEREG), KGRE (for example KGREG), KDRE (for exampleKDREV) are possible. Some other possible, but less preferred sequencesinclude for example DECKL and NVCEL. d) With both GLEW at positions44-47 and KERE or KQRE at positions 43-46. e) Often as KP or EP atpositions 83-84 of naturally occurring V_(HH) domains. f) In particular,but not exclusively, in combination with GLEW at positions 44-47. g)With the proviso that when positions 44-47 are GLEW, position 108 in(non-humanized) VHH sequences that also contain a W at 103. h) The GLEWgroup also contains GLEW-like sequences at positions 44-47, such as forexample GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLERand ELEW.

TABLE A-4 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring Nanobodies. For humanization of thesecombinations, reference is made to the specification. 11 37 44 45 47 8384 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R LK P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q LF L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group LV G L W K S W G Q M V G L W K P R G Q

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. TablesA-5-A-8 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1.)

In Tables A-5-A-8, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables A-5 to A-8 also contain data on the V_(HH)entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 1118 V_(HH) sequences(data kindly provided by David Lutje Hulsing and Prof. Theo Verrips ofUtrecht University). The values for the V_(HH) entropy and the V_(HH)variability provide a measure for the variability and degree ofconservation of amino acid residues between the 1118 V_(HH) sequencesanalyzed: low values (i.e. <1, such as <0.5) indicate that an amino acidresidue is highly conserved between the V_(HH) sequences (i.e. littlevariability). For example, the G at position 8 and the G at position 9have values for the V_(HH) entropy of 0.1 and 0 respectively, indicatingthat these residues are highly conserved and have little variability(and in case of position 9 is G in all 1118 sequences analysed), whereasfor residues that form part of the CDR's generally values of 1.5 or moreare found (data not shown). Note that (1) the amino acid residues listedin the second column of Tables A-5-A-8 are based on a bigger sample thanthe 1118 V_(HH) sequences that were analysed for determining the V_(HH)entropy and V_(HH) variability referred to in the last two columns; and(2) the data represented below support the hypothesis that the aminoacid residues at positions 27-30 and maybe even also at positions 93 and94 already form part of the CDR's (although the invention is not limitedto any specific hypothesis or explanation, and as mentioned above,herein the numbering according to Kabat is used). For a generalexplanation of sequence entropy, sequence variability and themethodology for determining the same, see Oliveira et al., PROTEINS:Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)Position Human V_(H)3 Camelid V_(HH)'s V_(HH) Ent. Var. 1 E, Q Q, A, E —— 2 V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E,D, Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R0.3 2 11 Hallmark residue: L, M, S, 0.8 2 V, W; preferably L 12 V, I V,A 0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 515 G G, D 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 119 R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.23 22 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 625 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R,L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, 1.7 11 G, R, F, Y 29 F,V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G N, S, E, G, A, D, M, T 1.8 11

TABLE A-6 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Position Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0.1 1 37Hallmark residue: F⁽¹⁾, H, I, L, 1.1 6 Y or V, preferably F⁽¹⁾ or Y 38 RR 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, TP, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, Q, R, 1.3 5 S, L; preferably G⁽²⁾,E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾. 45 Hallmark residue: L⁽²⁾,R⁽³⁾, C, I, L, 0.6 4 P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 46 E, V E, D, K,Q, V 0.4 2 47 Hallmark residue: W⁽²⁾, L⁽¹⁾ or 1.9 9 F⁽¹⁾, A, G, I, M, R,S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A,G A, S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)Position Human V_(H)3 Camelid V_(HH)'s V_(HH) Ent. Var. 66 R R 0.1 1 67F F, L, V 0.1 1 68 T T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A,F, T 0.3 4 71 R R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V0.5 4 73 N, D, G N, A, D, F, I, K, L, R, S, 1.2 9 T, V, Y 74 A, S A, D,G, N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K,R, S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A,F, G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 181 Q Q, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2 82a N, G N, D, G, H,S, T 0.8 4 82b S S, N, D, G, R, T 1 6 82c L L, P, V 0.1 2 83 Hallmarkresidue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, 0.9 7 Q or T; preferably K or R;most preferably K 84 Hallmark residue: P⁽⁵⁾, A, D, L, R, S, T, V; 0.7 6preferably P 85 E, G E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 388 A A, G, S 0.3 2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 191 Y, H Y, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H,K, N, R, S, 1.4 10 T, V, Y 94 K, R, T A, V, C, F, G, I, K, L, R, 1.6 9 Sor T

TABLE A-8 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)Position Human V_(H)3 Camelid V_(HH)'s V_(HH) Ent. Var. 103 Hallmarkresidue: W⁽⁴⁾, P⁽⁶⁾, 0.4 2 R⁽⁶⁾, S; preferably W 104 Hallmark residue: Gor 0.1 1 D; preferably G 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1 107T T, A, I 0.3 2 108 Hallmark residue: Q, L⁽⁷⁾ or R; 0.4 3 preferably Qor L⁽⁷⁾ 109 V V 0.1 1 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F0.3 1 113 S S, A, L, P, T 0.4 3

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a) the Hallmark residues are as defined herein;    and in which:-   b) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

and in which

-   a) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 1] [1] QVQLQESGGGXVQAGGSLRLSCAASG [26]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with the above amino acid sequence; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);    -    and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:-   b) FR2 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 2] [36] WXRQAPGKXXEXVA [49]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with the above amino acid sequence; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:-   c) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 3] [66] RFTISRDNAKNTVYLQMNSLXXEDTAVYYCAA [94]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with the above amino acid sequence; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:-   d) FR4 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 4] [103] XXQGTXVTVSS [113]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with the above amino acid sequence; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and in which:-   e) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein;    in which the Hallmark Residues are indicated by “X” and are as    defined hereinabove and in which the numbers between brackets refer    to the amino acid positions according to the Kabat numbering.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   a) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 5] [1] QVQLQESGGGLVQAGGSLRLSCAASG [26]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with the above amino acid sequence; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residue at position is as indicated in the        sequence above;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residue at position is as indicated in the        sequence above;        and in which:-   b) FR2 is chosen from the group consisting of the amino acid    sequences:

[SEQ ID NO: 6] [36] WFRQAPGKERELVA [49] [SEQ ID NO: 7][36] WFRQAPGKEREFVA [49] [SEQ ID NO: 8] [36] WFRQAPGKEREGA [49] [SEQ IDNO: 9] [36] WFRQAPGKQRELVA [49] [SEQ ID NO: 10] [36] WFRQAPGKQREFVA [49][SEQ ID NO: 11] [36] WYRQAPGKGLEWA [49]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with one of the above amino acid sequences; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;        and in which:-   c) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with the above amino acid sequence; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;

and in which:

-   d) FR4 is chosen from the group consisting of the amino acid    sequences:

[SEQ ID NO: 13] [103] WGQGTQVTVSS [113] [SEQ ID NO: 14][103] WGQGTLVTVSS [113]

-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with one of the above amino acid sequence; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;

and in which:

-   e) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which: and in which

-   a) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 5] [1] QVQLQESGGGLVQAGGSLRLSCAASG [26]

-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residue at position is as indicated in the        sequence above;

and in which:

-   b) FR2 is chosen from the group consisting of the amino acid    sequences:

[SEQ ID NO: 6] [36] WFRQAPGKERELVA [49] [SEQ ID NO: 7][36] WFRQAPGKEREFVA [49] [SEQ ID NO: 8] [36] WFRQAPGKEREGA [49] [SEQ IDNO: 9] [36] WFRQAPGKQRELVA [49] [SEQ ID NO: 10] [36] WFRQAPGKQREFVA [49]

-    and/or from the group consisting of amino acid sequences that have    2 or only 1 “amino acid difference(s)” (as defined herein) with one    of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;        and in which:-   c) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94]

-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;        and in which:-   d) FR4 is chosen from the group consisting of the amino acid    sequences:

[SEQ ID NO: 13] [103] WGQGTQVTVSS [113] [SEQ ID NO: 14][103] WGQGTLVTVSS [113]

-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;        and in which:-   e) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a) FR1 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 5] [1] QVQLQESGGGLVQAGGSLRLSCAASG [26]

-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residue at position is as indicated in the        sequence above;        and in which:-   b) FR2 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 11] [36] WYRQAPGKGLEWA [49]

-    and/or from the group consisting of amino acid sequences that have    2 or only 1 “amino acid difference(s)” (as defined herein) with one    of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in each of the sequences above;        and in which:-   c) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94]

-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 83 and 84 are as        indicated in each of the sequences above;        and in which:-   d) FR4 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 13] [103] WGQGTQVTVSS [113]

-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    one of the above amino acid sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s); and    -   iii) the Hallmark residues at positions 103, 104 and 108 are as        indicated in each of the sequences above;        and in which:-   e) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

Some other framework sequences that can be present in the Nanobodies ofthe invention can be found in the European patent EP 656 946 mentionedabove (see for example also the granted U.S. Pat. No. 5,759,808),

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

and in which

-   a) FR1 is chosen from the group consisting of the amino acid    sequences of SEQ ID NO's: 42-92, or from the group consisting of    amino acid sequences that have at least 80%, preferably at least    90%, more preferably at least 95%, even more preferably at least 99%    sequence identity (as defined herein) with at least one of said FR1    sequences; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR1 sequence; and    -   iii) the Hallmark residue at position is as indicated in said        FR1 sequence;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    at least one of said FR1 sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-5; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR1 sequence; and    -   iii) the Hallmark residue at position is as indicated in said        FR1 sequence;

and in which:

-   b) FR2 is chosen from the group consisting of the amino acid    sequences of SEQ ID NO's: 144-194,-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with at least one of said FR2 sequences; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR2 sequence; and-   iii) the Hallmark residues at positions 37, 44, 45 and 47 are as    indicated in said FR2 sequence;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    at least one of said FR2 sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-6; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR2 sequence; and    -   iii) the Hallmark residues at positions 37, 44, 45 and 47 are as        indicated in said FR2 sequence;        and in which:-   c) FR3 is chosen from the group consisting of the amino acid    sequences of SEQ ID NO's: 246-296, or from the group consisting of    amino acid sequences that have at least 80%, preferably at least    90%, more preferably at least 95%, even more preferably at least 99%    sequence identity (as defined herein) with at least one of said FR3    sequences; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR3 sequence; and    -   iii) the Hallmark residues at positions 83 and 84 are as        indicated in said FR3 sequence;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    at least one of said FR3 sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-7; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR3 sequence; and    -   iii) the Hallmark residues at positions 83 and 84 are as        indicated in said FR3 sequence;        and in which:-   d) FR4 is chosen from the group consisting of the amino acid    sequences of SEQ ID NO's: 348-398,-    or from the group consisting of amino acid sequences that have at    least 80%, preferably at least 90%, more preferably at least 95%,    even more preferably at least 99% sequence identity (as defined    herein) with at least one of said FR4 sequences; in which    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR4 sequence; and    -   iii) the Hallmark residues at positions 103, 104 and 108 are as        indicated in said FR3 sequence;-    and/or from the group consisting of amino acid sequences that have    3, 2 or only 1 “amino acid difference(s)” (as defined herein) with    at least one of said FR4 sequences, in which:    -   i) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Table A-8; and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to said FR4 sequence; and    -   iii) the Hallmark residues at positions 103, 104 and 108 are as        indicated in said FR4 sequence;        and in which:-   e) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred embodiments herein, and    are more preferably as defined according to one of the more    preferred embodiments herein.

Some particularly preferred Nanobodies of the invention can be chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:399-449, or from the group consisting of amino acid sequences that haveat least 80%, preferably at least 90%, more preferably at least 95%,even more preferably at least 99% sequence identity (as defined herein)with at least one of said amino acid sequences; in which

-   a) the Hallmark residues can be as indicated in Table A-3 above;-   b) any amino acid substitution at any position other than a Hallmark    position is preferably either a conservative amino acid substitution    (as defined herein) and/or an amino acid substitution as defined in    Tables A-5-A-8; and/or-   c) said amino acid sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the above amino acid sequence(s).

Some even more particularly preferred Nanobodies of the invention can bechosen from the group consisting of the amino acid sequences of SEQ IDNO's: 399-449, or from the group consisting of amino acid sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity (as definedherein) with at least one of said amino acid sequences; in which

-   a) the Hallmark residues are as indicated in the pertinent sequence    from SEQ ID NO's: 399-449;-   b) any amino acid substitution at any position other than a Hallmark    position is preferably either a conservative amino acid substitution    (as defined herein) and/or an amino acid substitution as defined in    Tables A-5-A-8; and/or-   c) said amino acid sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the pertinent sequence chosen from SEQ ID NO's: 399-449.

Some of the most preferred Nanobodies of the invention against the IL-6receptor can be chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 399-449.

Preferably, the CDR sequences and FR sequences in the Nanobodies of theinvention are such that the Nanobody of the invention binds to the IL-6receptor, with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to IL-6R with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to IL-6R with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to IL-6R with a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to IL-6R with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to IL-6R will become clear from thefurther description and examples herein.

According to one non-limiting aspect of the invention, a Nanobody may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a Nanobody may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a Nanobody will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at at least one of the Hallmarkresidues in FR2 and/or FR4 (again, (including those at positions 108,103 and/or 45).

Also, a humanized Nanobody of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring VHH domain.More specifically, according to one non-limiting aspect of theinvention, a humanized Nanobody may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring VHH domain. Usually, a humanizedNanobody will have at least one such amino acid difference with anaturally occurring VHH domain in at least one of FR2 and/or FR4, and inparticular at at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the Nanobodies of the invention as defined herein, and inparticular analogs of the Nanobodies of SEQ ID NO's: 399-449. Thus,according to one embodiment of the invention, the term “Nanobody of theinvention” in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the Nanobodies of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables A-5-A-8for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the Nanobody of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the Nanobody of theinvention (i.e. to the extent that the Nanobody is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express theNanobody or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5-A-8 above, some amino acid residues inthe framework regions are more conserved than others. Generally,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. Also, generally, amino acid substitutions arepreferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to the IL-6 receptor,with affinity (suitably measured and/or expressed as a K_(D)-value(actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rateand/or a k_(off)-rate, or alternatively as an IC₅₀ value, as furtherdescribed herein) that is as defined herein; as well as compounds andconstructs, and in particular proteins and polypeptides, that compriseat least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to IL-6R with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to IL-6R with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 103 M⁻¹ s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 104 M⁻¹ s⁻¹ and 107 M⁻¹ s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to IL-6R with a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to IL-6R with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to IL-6R will become clear from thefurther description and examples herein.

The analogs are preferably also such that they retain the favourableproperties the Nanobodies, as described herein.

Also, according to one preferred embodiment, the analogs have a degreeof sequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the Nanobodies of SEQ ID NOs 399-449.

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred embodiments definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and R at position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the Nanobodies of the inventioncomprise Nanobodies that have been humanized (i.e. compared to thesequence of a naturally occurring Nanobody of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparision between the sequence of a Nanobody and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized Nanobodies still retain the favourable properties ofNanobodies as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the inventionmay become more “human-like”, while still retaining the favorableproperties of the Nanobodies of the invention as described herein. As aresult, such humanized Nanobodies may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The Nanobodies of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for Nanobodies of the “P,R,S-103group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a camelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized Nanobodies has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se. For example, theanalogs can be obtained by providing a nucleic acid that encodes anaturally occurring V_(HH) domain, changing the codons for the one ormore amino acid residues that are to be substituted into the codons forthe corresponding desired amino acid residues (e.g. by site-directedmutagenesis or by PCR using suitable mismatch primers), expressing thenucleic acid/nucleotide sequence thus obtained in a suitable host orexpression system; and optionally isolating and/or purifying the analogthus obtained to provide said analog in essentially isolated form (e.g.as further described herein). This can generally be performed usingmethods and techniques known per se, which will be clear to the skilledperson, for example from the handbooks and references cited herein, thebackground art cited herein and/or from the further description herein.Alternatively, a nucleic acid encoding the desired analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can then be expressed as described herein. Yetanother technique may involve combining one or more naturally occurringand/or synthetic nucleic acid sequences each encoding a part of thedesired analog, and then expressing the combined nucleic acid sequenceas described herein. Also, the analogs can be provided using chemicalsynthesis of the pertinent amino acid sequence using techniques forpeptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that theNanobodies of the invention (including their analogs) can be designedand/or prepared starting from human V_(H) sequences (i.e. amino acidsequences or the corresponding nucleotide sequences), such as forexample from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e.by introducing one or more camelizing substitutions (i.e. changing oneor more amino acid residues in the amino acid sequence of said humanV_(H) domain into the amino acid residues that occur at thecorresponding position in a V_(HH) domain), so as to provide thesequence of a Nanobody of the invention and/or so as to confer thefavourable properties of a Nanobody to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using an amino acidsequence and/or nucleotide sequence for a human V_(H) domain as astarting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables A-5-A-8. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of Nanobodies are obtained or improved (i.e. compared to theoriginal V_(H) domain).

Generally, however, such camelizing substitutions are preferably suchthat the resulting an amino acid sequence at least contains (a) a Q atposition 108; and/or (b) a charged amino acid or a cysteine residue atposition 45 and preferably also an E at position 44, and more preferablyE at position 44 and R at position 45; and/or (c) P, R or S at position103; and optionally one or more further camelizing substitutions. Morepreferably, the camelizing substitutions are such that they result in aNanobody of the invention and/or in an analog thereof (as definedherein), such as in a humanized analog and/or preferably in an analogthat is as defined in the preceding paragraphs.

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the Nanobodies of the invention asdefined herein, and in particular parts or fragments of the Nanobodiesof SEQ ID NO's 399-449. Thus, according to one embodiment of theinvention, the term “Nanobody of the invention” in its broadest sensealso covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention(including analogs thereof) have amino acid sequences in which, comparedto the amino acid sequence of the corresponding full length Nanobody ofthe invention (or analog thereof), one or more of the amino acidresidues at the N-terminal end, one or more amino acid residues at theC-terminal end, one or more contiguous internal amino acid residues, orany combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to theIL-6 receptor, with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to IL-6R with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to IL-6R with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more        preferably between 10⁴ M⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to IL-6R with a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁴ s⁻¹, such as        between 10⁻⁴ M⁻¹s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to IL-6R with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to IL-6R will become clear from thefurther description and examples herein.

The affinity of the analog against the IL-6 receptor, can be determinedin a manner known per se, for example using the assay described herein.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length Nanobody of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limitingembodiment, such a part or fragment comprises at least CDR3, such asFR3, CDR3 and FR4 of the corresponding full length Nanobody of theinvention, i.e. as for example described in the Internationalapplication WO 03/050531 (Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different Nanobodiesof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of a Nanobody of the invention with one or more partsor fragments of a human V_(H) domain.

According to one preferred embodiment, the parts or fragments have adegree of sequence identity of at least 50%, preferably at least 60%,more preferably at least 70%, even more preferably at least 80%, such asat least 90%, 95% or 99% or more with one of the Nanobodies of SEQ IDNOs: 399-449.

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized Nanobody of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized Nanobody of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.genzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that that increase the half-life, the solubilityand/or the absorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked or0-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²p, ³⁵S,¹⁴C, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metals chelates ormetallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to the IL-6receptor, with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to IL-6R with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to IL-6R with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to IL-6R with a k_(off) rate between 1s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to IL-6R with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to IL-6R will become clear from thefurther description and examples herein.

The affinity of a derivative of a Nanobody of the invention against theIL-6 receptor, can be determined in a manner known per se, for exampleusing the assay described herein.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a Nanobody of theinvention or corresponds to the amino acid sequence of a Nanobody of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   a) can comprise an N-terminal Met residue, for example as result of    expression in a heterologous host cell or host organism.-   b) may form a signal sequence or leader sequence that directs    secretion of the Nanobody from a host cell upon synthesis. Suitable    secretory leader peptides will be clear to the skilled person, and    may be as further described herein. Usually, such a leader sequence    will be linked to the N-terminus of the Nanobody, although the    invention in its broadest sense is not limited thereto;-   c) may form a sequence or signal that allows the Nanobody to be    directed towards and/or to penetrate or enter into specific organs,    tissues, cells, or parts or compartments of cells, and/or that    allows the Nanobody to penetrate or cross a biological barrier such    as a cell membrane, a cell layer such as a layer of epithelial    cells, a tumor including solid tumors, or the blood-brain-barrier.    Examples of such amino acid sequences will be clear to the skilled    person. Some non-limiting examples are the small peptide vectors    (“Pep-trans vectors”) described in WO 03/026700 and in Temsamani et    al., Expert Opin. Biol. Ther., 1, 773 (2001); Temsamani and Vidal,    Drug Discov. Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp.    Ther., 296, 124-131 (2001), and the membrane translocator sequence    described by Zhao et al., Apoptosis, 8, 631-637 (2003). C-terminal    and N-terminal amino acid sequences for intracellular targeting of    antibody fragments are for example described by Cardinale et al.,    Methods, 34, 171 (2004). Other suitable techniques for intracellular    targeting involve the expression and/or use of so-called    “intrabodies” comprising a Nanobody of the invention, as mentioned    below;-   d) may form a “tag”, for example an amino acid sequence or residue    that allows or facilitates the purification of the Nanobody, for    example using affinity techniques directed against said sequence or    residue. Thereafter, said sequence or residue may be removed (e.g.    by chemical or enzymatical cleavage) to provide the Nanobody    sequence (for this purpose, the tag may optionally be linked to the    Nanobody sequence via a cleavable linker sequence or contain a    cleavable motif). Some preferred, but non-limiting examples of such    residues are multiple histidine residues, glutatione residues and a    myc-tag such as AAAEQKLISEEDLNGAA [SEQ ID NO:31];-   e) may be one or more amino acid residues that have been    functionalized and/or that can serve as a site for attachment of    functional groups. Suitable amino acid residues and functional    groups will be clear to the skilled person and include, but are not    limited to, the amino acid residues and functional groups mentioned    herein for the derivatives of the Nanobodies of the invention.

According to another embodiment, a polypeptide of the inventioncomprises a Nanobody of the invention, which is fused at its aminoterminal end, at its carboxy terminal end, or both at its amino terminalend and at its carboxy terminal end to at least one further amino acidsequence, i.e. so as to provide a fusion protein comprising saidNanobody of the invention and the one or more further amino acidsequences. Such a fusion will also be referred to herein as a “Nanobodyfusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005),

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the Nanobody of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

The further amino acid sequence may also provide a second binding site,which binding site may be directed against any desired protein,polypeptide, antigen, antigenic determinant or epitope (including butnot limited to the same protein, polypeptide, antigen, antigenicdeterminant or epitope against which the Nanobody of the invention isdirected, or a different protein, polypeptide, antigen, antigenicdeterminant or epitope). For example, the further amino acid sequencemay provide a second binding site that is directed against a serumprotein (such as, for example, human serum albumin or another serumprotein such as IgG), so as to provide increased half-life in serum.Reference is for example made to EP 0 368 684, WO 91/01743, WO 01/45746and WO 04/003019 (in which various serum proteins are mentioned), theInternational application by Ablynx N. V. entitled “Nanobodies againstamyloid-beta and polypeptides comprising the same for the treatment ofdegenerative neural diseases such as Alzheimer's disease” (in whichvarious other proteins are mentioned), as well as to Harmsen et al.,Vaccine, 23 (41); 4926-42, 2005.

According to another embodiment, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such Nanobodies, compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise Nanobodies sequencesor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);amino acid sequences of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin. Reference is for example made to the US provisional applicationby Ablynx N. V. entitled “Immunoglobulin domains with multiple bindingsites” filed on Nov. 27, 2006); or polypeptides of the invention thatcomprise at least one Nanobody of the invention that is linked to atleast one moiety (and in particular at least one amino acid sequence)that increases the half-life of the Nanobody of the invention. Examplesof polypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more Nanobodies of theinvention are suitable linked to one or more serum proteins or fragmentsthereof (such as serum albumin or suitable fragments thereof) or to oneor more binding units that can bind to serum proteins (such as, forexample, Nanobodies or (single) domain antibodies that can bind to serumproteins such as serum albumin, serum immunoglobulins such as IgG, ortransferrine); polypeptides in which a Nanobody of the invention islinked to an Fc portion (such as a human Fc) or a suitable part orfragment thereof; or polypeptides in which the one or more Nanobodies ofthe invention are suitable linked to one or more small proteins orpeptides that can bind to serum proteins (such as, without limitation,the proteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489).

Generally, the Nanobodies of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the corresponding aminoacid sequence of the invention per se. For example, the Nanobodies,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), at preferably at least 9 days (such asabout 9 to 14 days), more preferably at least about 10 days (such asabout 10 to 15 days), or at least about 11 days (such as about 11 to 16days), more preferably at least about 12 days (such as about 12 to 18days or more), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) Nanobodies of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) amino acid sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more amino acid sequences thatallow the resulting polypeptides of the invention to cross the bloodbrain barrier may be one or more (such as two and preferably one)Nanobodies, such as the Nanobodies described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137). According to the invention,the Nanobody of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the Nanobody of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to the U.S. provisional application 60/788,256 ofAblynx N. V. entitled “Albumin derived amino acid sequence, use thereoffor increasing the half-life of therapeutic proteins and of othertherapeutic proteins and entities, and constructs comprising the same”filed on Mar. 31, 2006.

Some preferred, but non-limiting, examples of fusions of anti-IL6RNanobodies to human serum albumin are given in SEQ ID NO's: 603-608.

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such amino acidsequences for example include the Nanobodies described below, as well asthe small peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, as well as to EP 0 368 684, as well as to the following theU.S. provisional applications 60/843,349, 60/850,774, 60/850,775 byAblynx N. V. mentioned herein.

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and that amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againsee for example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N. V. entitled “Serum albumin bindingproteins with long half-lives” filed on Sep. 8, 2006); amino acidsequences against human serum albumin that are cross-reactive with serumalbumin from at least one species of mammal, and in particular with atleast one species of primate (such as, without limitation, monkeys fromthe genus Macaca (such as, and in particular, cynomologus monkeys(Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon(Papio ursinus), reference is again made to the U.S. provisionalapplication 60/843,349); amino acid sequences that can bind to serumalbumin in a pH independent manner (see for example the U.S. provisionalapplication 60/850,774 by Ablynx N. V. entitled “Amino acid sequencesthat bind to serum proteins in a manner that is essentially independentof the pH, compounds comprising the same, and uses thereof”, filed onOct. 11, 2006) and/or amino acid sequences that are conditional binders(see for example the U.S. provisional application 60/850,775 by AblynxN. V. entitled “Amino acid sequences that bind to a desired molecule ina conditional manner”, filed on Oct. 11, 2006).

The at least one Nanobody may also be linked to one or more (preferablyhuman) CH₁, CH₂ and/or CH₃ domains, optionally via a linker sequence.For instance, a Nanobody linked to a suitable CH₁ domain could forexample be used—together with suitable light chains—to generate antibodyfragments/structures analogous to conventional Fab fragments or F(ab′)2fragments, but in which one or (in case of an F(ab′)2 fragment) one orboth of the conventional V_(H) domains have been replaced by a Nanobodyof the invention. Also, two Nanobodies could be linked to a CH3 domain(optionally via a linker) to provide a construct with increasedhalf-life in vivo.

According to one specific embodiment of a polypeptide of the invention,one or more Nanobodies of the invention may be linked to one or moreantibody parts, fragments or domains that confer one or more effectorfunctions to the polypeptide of the invention and/or may confer theability to bind to one or more Fc receptors. For example, for thispurpose, and without being limited thereto, the one or more furtheramino acid sequences may comprise one or more CH₂ and/or CH₃ domains ofan antibody, such as from a heavy chain antibody (as described herein)and more preferably from a conventional human 4-chain antibody; and/ormay form (part of) and Fc region, for example from IgG, from IgE or fromanother human Ig. For example, WO 94/04678 describes heavy chainantibodies comprising a Camelid V_(HH) domain or a humanized derivativethereof (i.e. a Nanobody), in which the Camelidae CH₂ and/or CH₃ domainhave been replaced by human CH₂ and CH₃ domains, so as to provide animmunoglobulin that consists of 2 heavy chains each comprising aNanobody and human CH2 and CH3 domains (but no CH1 domain), whichimmunoglobulin has the effector function provided by the CH2 and CH3domains and which immunoglobulin can function without the presence ofany light chains. Other amino acid sequences that can be suitably linkedto the Nanobodies of the invention so as to provide an effector functionwill be clear to the skilled person, and may be chosen on the basis ofthe desired effector function(s). Reference is for example made to WO04/058820, WO 99/42077 and WO 05/017148, as well as the review byHolliger and Hudson, supra. Coupling of a Nanobody of the invention toan Fc portion may also lead to an increased half-life, compared to thecorresponding Nanobody of the invention. For some applications, the useof an Fc portion and/or of constant domains (i.e. CH₂ and/or CH₃domains) that confer increased half-life without any biologicallysignificant effector function may also be suitable or even preferred.Other suitable constructs comprising one or more Nanobodies and one ormore constant domains with increased half-life in vivo will be clear tothe skilled person, and may for example comprise two Nanobodies linkedto a CH3 domain, optionally via a linker sequence. Generally, any fusionprotein or derivatives with increased half-life will preferably have amolecular weight of more than 50 kD, the cut-off value for renalabsorption.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, the “Peptrans” vectors mentioned above, the sequencesdescribed by Cardinale et al. and the amino acid sequences and antibodyfragments known per se that can be used to express or produce theNanobodies and polypeptides of the invention as so-called “intrabodies”,for example as described in WO 94/02610, WO 95/22618, US-A-7004940, WO03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting embodiment, said one ormore further amino acid sequences comprise at least one furtherNanobody, so as to provide a polypeptide of the invention that comprisesat least two, such as three, four, five or more Nanobodies, in whichsaid Nanobodies may optionally be linked via one or more linkersequences (as defined herein). Polypeptides of the invention thatcomprise two or more Nanobodies, of which at least one is a Nanobody ofthe invention, will also be referred to herein as “multivalent”polypeptides of the invention, and the Nanobodies present in suchpolypeptides will also be referred to herein as being in a “multivalentformat”. For example a “bivalent” polypeptide of the invention comprisestwo Nanobodies, optionally linked via a linker sequence, whereas a“trivalent” polypeptide of the invention comprises three Nanobodies,optionally linked via two linker sequences; etc.; in which at least oneof the Nanobodies present in the polypeptide, and up to all of theNanobodies present in the polypeptide, is/are a Nanobody of theinvention.

In a multivalent polypeptide of the invention, the two or moreNanobodies may be the same or different, and may be directed against thesame antigen or antigenic determinant (for example against the samepart(s) or epitope(s) or against different parts or epitopes) or mayalternatively be directed against different antigens or antigenicdeterminants; or any suitable combination thereof. For example, abivalent polypeptide of the invention may comprise (a) two identicalNanobodies; (b) a first Nanobody directed against a first antigenicdeterminant of a protein or antigen and a second Nanobody directedagainst the same antigenic determinant of said protein or antigen whichis different from the first Nanobody; (c) a first Nanobody directedagainst a first antigenic determinant of a protein or antigen and asecond Nanobody directed against another antigenic determinant of saidprotein or antigen; or (d) a first Nanobody directed against a firstprotein or antigen and a second Nanobody directed against a secondprotein or antigen (i.e. different from said first antigen). Similarly,a trivalent polypeptide of the invention may, for example and withoutbeing limited thereto. comprise (a) three identical Nanobodies; (b) twoidentical Nanobody against a first antigenic determinant of an antigenand a third Nanobody directed against a different antigenic determinantof the same antigen; (c) two identical Nanobody against a firstantigenic determinant of an antigen and a third Nanobody directedagainst a second antigen different from said first antigen; (d) a firstNanobody directed against a first antigenic determinant of a firstantigen, a second Nanobody directed against a second antigenicdeterminant of said first antigen and a third Nanobody directed againsta second antigen different from said first antigen; or (e) a firstNanobody directed against a first antigen, a second Nanobody directedagainst a second antigen different from said first antigen, and a thirdNanobody directed against a third antigen different from said first andsecond antigen.

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen (i.e.against the IL-6 receptor) and at least one Nanobody is directed againsta second antigen (i.e. different from the IL-6 receptor), will also bereferred to as “multispecific” polypeptides of the invention, and theNanobodies present in such polypeptides will also be referred to hereinas being in a “multispecific format”. Thus, for example, a “bispecific”polypeptide of the invention is a polypeptide that comprises at leastone Nanobody directed against a first antigen (i.e. the IL-6 receptor)and at least one further Nanobody directed against a second antigen(i.e. different from the IL-6 receptor), whereas a “trispecific”polypeptide of the invention is a polypeptide that comprises at leastone Nanobody directed against a first antigen (i.e. the IL-6 receptor),at least one further Nanobody directed against a second antigen (i.e.different from the IL-6 receptor) and at least one further Nanobodydirected against a third antigen (i.e. different from both the IL-6receptor, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against the IL-6 receptor,and a second Nanobody directed against a second antigen, in which saidfirst and second Nanobody may optionally be linked via a linker sequence(as defined herein); whereas a trispecific polypeptide of the inventionin its simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first Nanobody directed against the IL-6receptor, a second Nanobody directed against a second antigen and athird Nanobody directed against a third antigen, in which said first,second and third Nanobody may optionally be linked via one or more, andin particular one and more in particular two, linker sequences.

Some preferred, but non-limiting examples of bivalent bispecificpolypeptides of the invention are given in SEQ ID NO's: 450-471 and559-602. Some preferred, but non-limiting examples of trivalentbispecific polypeptides of the invention are given in SEQ ID NO's:478-558.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one Nanobody againstthe IL-6 receptor, and any number of Nanobodies directed against one ormore antigens different from the IL-6 receptor.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity for the IL-6receptor, or against the one or more other antigens), said order orarrangement is usually not critical and may be suitably chosen by theskilled person, optionally after some limited routine experiments basedon the disclosure herein. Thus, when reference is made to a specificmultivalent or multispecific polypeptide of the invention, it should benoted that this encompasses any order or arrangements of the relevantNanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that thepolypeptides of the invention contain two or more Nanobodies and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptidee of the inventioncan be found in the applications by Ablynx N. V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one Nanobody of the invention and atleast one Nanobody that provides for an increased half-life. Somepreferred, but non-limiting examples of such Nanobodies includeNanobodies directed against serum proteins, such as human serum albumin,thyroxine-binding protein, (human) transferrin, fibrinogen, animmunoglobulin such as IgG, IgE or IgM, or one of the other serumproteins listed in WO 04/003019.

For example, for experiments in mice, Nanobodies against mouse serumalbumin (MSA) can be used, whereas for pharmaceutical use, Nanobodiesagainst human serum albumin can be used.

Another embodiment of the present invention is a polypeptide constructas described above wherein said at least one (human) serum protein isany of (human) serum albumin, (human) serum immunoglobulins, (human)thyroxine-binding protein, (human) transferrin, (human) fibrinogen, etc.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNanobodies of the invention, at least one Nanobody against human serumalbumin. Although these Nanobodies against human serum albumin may be asgenerally described in the applications by Ablynx N. V. cited above (seefor example W04/062551), according to a particularly preferred, butnon-limiting embodiment, said Nanobody against human serum albuminconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

i) CDR1 is an amino acid sequence chosen from the group consisting of:

[SEQ ID NO: 15] FGMS [SEQ ID NO: 16] LNLMG [SEQ ID NO: 17] INLLG [SEQ IDNO: 18] NYWMY;

and/or from the group consisting of amino acid sequences that have 2 oronly 1 “amino acid difference(s)” (as defined herein) with one of theabove amino acid sequences, in which:

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences;

and in which:

ii) CDR2 is an amino acid sequence chosen from the group consisting of:

[SEQ ID NO: 19] SISGSGSDTLYADSVKG [SEQ ID NO: 20] TITVGDSTNYADSVKG [SEQID NO: 21] TITVGDSTSYADSVKG [SEQ ID NO: 22] SINGRGDDTRYADSVKG [SEQ IDNO: 23] AISADSSTKNYADSVKG [SEQ ID NO: 24] AISADSSDKRYADSVKG [SEQ ID NO:25] RISTGGGYSYYADSVKG

or from the group consisting of amino acid sequences that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with oneof the above amino acid sequences; in which

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences;

and/or from the group consisting of amino acid sequences that have 3, 2or only 1 “amino acid difference(s)” (as defined herein) with one of theabove amino acid sequences, in which:

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences;

and in which:

iii) CDR3 is an amino acid sequence chosen from the group consisting of:

[SEQ ID NO: 26] DREAQVDTLDFDY

or from the group consisting of amino acid sequences that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with oneof the above amino acid sequences; in which

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences;

and/or from the group consisting of amino acid sequences that have 3, 2or only 1 “amino acid difference(s)” (as defined herein) with one of theabove amino acid sequences, in which:

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences;

or from the group consisting of:

[SEQ ID NO: 27] GGSLSR [SEQ ID NO: 28] RRTWHSEL [SEQ ID NO: 29] GRSVSRS[SEQ ID NO: 30] GRGSP

and/or from the group consisting of amino acid sequences that have 3, 2or only 1 “amino acid difference(s)” (as defined herein) with one of theabove amino acid sequences, in which:

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences.

In another aspect, the invention relates to a Nanobody against humanserum albumin, which consist of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), which is chosen from the group consisting of Nanobodieswith the one of the following combinations of CDR1, CDR2 and CDR3,respectively:

CDR1: SFGMS; CDR2: SISGSGSDTLYADSVKG; CDR3: GGSLSR; CDR1: LNLMG; CDR2:TITVGDSTNYADSVKG; CDR3: RRTWHSEL; CDR1: INLLG; CDR2: TITVGDSTSYADSVKG;CDR3: RRTWHSEL; CDR1: SFGMS; CDR2: SINGRGDDTRYADSVKG; CDR3: GRSVSRS;CDR1: SFGMS; CDR2: AISADSSDKRYADSVKG; CDR3: GRGSP; CDR1: SFGMS; CDR2:AISADSSDKRYADSVKG; CDR3: GRGSP; CDR1: NYWMY; CDR2: RISTGGGYSYYADSVKG;CDR3: DREAQVDTLDFDY.

In the Nanobodies of the invention that comprise the combinations ofCDR's mentioned above, each CDR can be replaced by a CDR chosen from thegroup consisting of amino acid sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with thementioned CDR's; in which

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences;

and/or chosen from the group consisting of amino acid sequences thathave 3, 2 or only 1 (as indicated in the preceding paragraph) “aminoacid difference(s)” (as defined herein) with the mentioned CDR(s) one ofthe above amino acid sequences, in which:

(1) any amino acid substitution is preferably a conservative amino acidsubstitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acidsubstitutions, and no amino acid deletions or insertions, compared tothe above amino acid sequences.

However, of the Nanobodies of the invention that comprise thecombinations of CDR's mentioned above, Nanobodies comprising one or moreof the CDR's listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's listed above are more particularlypreferred; and Nanobodies comprising three of the CDR's listed above aremost particularly preferred.

In these Nanobodies against human serum albumin, the Framework regionsFR1 to FR4 are preferably as defined hereinabove for the Nanobodies ofthe invention.

Some preferred, but non-limiting examples of Nanobodies directed againsthuman serum albumin that can be used in the polypeptides of theinvention are listed in Table A-9 below. ALB-8 is a humanized version ofALB-1.

TABLE A-9 Preferred, but non-limiting examples of albumin-bindingNanobodies <Name, SEQ ID #; PRT (protein); -> Sequence <PMP 6A6(ALB-1),SEQ ID NO: 32; PRT; ->AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGG SLSRSSQGTQVTVSS<ALB-8(humanized ALB-1), SEQ ID NO: 33; PRT; ->EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSS <PMP6A8(ALB-2), SEQ ID NO: 34; PRT; ->AVQLVESGGGLVQGGGSLRLACAASERIFDLNLMGWYRQGPGNERELVATCITVGDSTNYADSVKGRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIRR TWHSELWGQGTQVTVSS

Some preferred, but non-limiting examples of polypeptides of theinvention that comprise at least one Nanobody against IL-6R and at leastone Nanobody that provides for increased half-life are given in SEQ IDNO's 478 to 602.

Generally, any derivatives and/or polypeptides of the invention withincreased half-life (for example pegylated Nanobodies or polypeptides ofthe invention, multispecific Nanobodies directed against the IL-6receptor and (human) serum albumin, or Nanobodies fused to an Fcportion, all as described herein) have a half-life that is at least 1.5times, preferably at least 2 times, such as at least 5 times, forexample at least 10 times or more than 20 times, the half-life of thecorresponding Nanobody of the invention.

Also, any derivatives or polypeptides of the invention with an increasehalf-life preferably have a half-life of more than 1 hour, preferablymore than 2 hours, more preferably of more than 6 hours, such as of morethan 12 hours, and for example of about one day, two days, one week, twoweeks or three weeks, and preferably no more than 2 months, although thelatter may be less critical.

Half-life can generally be defined as the time taken for the serumconcentration of the polypeptide to be reduce by 50%, in vivo, forexample due to degradation of the ligand and/or clearance orsequestration of the ligand by natural mechanisms. Methods forpharmacokinetic analysis and determination of half-life are familiar tothose skilled in the art. Details may be found in Kenneth, A et al:Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and inPeters et al, Pharmacokinete analysis: A Practical Approach (1996).Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron,published by Marcel Dekker, 2 nd Rev. ex edition (1982).

According to one aspect of the invention the polypeptides are capable ofbinding to one or more molecules which can increase the half-life of thepolypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

Another preferred, but non-limiting example of a multispecificpolypeptide of the invention comprises at least one Nanobody of theinvention and at least one Nanobody that directs the polypeptide of theinvention towards, and/or that allows the polypeptide of the inventionto penetrate or to enter into specific organs, tissues, cells, or partsor compartments of cells, and/or that allows the Nanobody to penetrateor cross a biological barrier such as a cell membrane, a cell layer suchas a layer of epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Examples of such Nanobodies include Nanobodies thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445, of which FC44 (SEQ ID NO35) and FC5 (SEQ ID NO: 36) are preferred examples.

TABLE A-10 Sequence listing of FC44 and FC5 <Name, SEQ ID #; PRT(protein); -> Sequence < FC44, SEQ ID NO: 35; PRT; ->EVQLQASGGGLVQAGGSLRLSCSASVRTFSIYAMGWFRQAPGKEREFVAGINRSGDVTKYADFVKGRFSISRDNAKNMVYLQMNSLKPEDTALYYCAATWAYDTVGALTSGYNFWGQGTQVTVSS < FC5, SEQ ID NO: 36; PRT; ->EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWGKGTQVTVSS

In the polypeptides of the invention, the one or more Nanobodies and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077, hinge-likeregions such as the hinge regions of naturally occurring heavy chainantibodies or similar sequences (such as described in WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers mentioned in Table A-11, of which AAA, GS-7and GS-9 are particularly preferred.

TABLE A-11 Sequence listing of linkers <Name, SEQ ID #; PRT (protein);-> Sequence < GS30, SEQ ID NO: 37; PRT; ->GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS < GS15, SEQ ID NO: 38; PRT; ->GGGGSGGGGSGGGGS < GS9, SEQ ID NO: 39; PRT; -> GGGGSGGGS < GS7, SEQ IDNO: 40; PRT; -> SGGSGGS < Llama upper long hinge region, SEQ ID NO: 41;PRT; -> EPKTPKPQPAAA

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for The IL-6 receptor, or for one or more of theother antigens. Based on the disclosure herein, the skilled person willbe able to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

For example, in multivalent polypeptides of the invention that compriseNanobodies directed against a multimeric antigen (such as a multimericreceptor or other protein), the length and flexibility of the linker arepreferably such that it allows each Nanobody of the invention present inthe polypeptide to bind to the antigenic determinant on each of thesubunits of the multimer. Similarly, in a multispecific polypeptide ofthe invention that comprises Nanobodies directed against two or moredifferent antigenic determinants on the same antigen (for exampleagainst different epitopes of an antigen and/or against differentsubunits of a multimeric receptor, channel or protein), the length andflexibility of the linker are preferably such that it allows eachNanobody to bind to its intended antigenic determinant. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 above) can provide improved hydrophilicproperties, whereas linkers that form or contain small epitopes or tagscan be used for the purposes of detection, identification and/orpurification. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thererto. For example, when a polypeptideof the invention comprises three of more Nanobodies, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a Nanobody, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedhereinabove).

According to one embodiment of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the Nanobodies and polypetides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments). Some preferred, butnon-limiting methods for preparing the amino acid sequences, Nanobodies,polypeptides and nucleic acids include the methods and techniquesdescribed herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, Nanobody and/or a polypeptide ofthe invention generally comprises the steps of:

-   -   the expression, in a suitable host cell or host organism (also        referred to herein as a “host of the invention”) or in another        suitable expression system of a nucleic acid that encodes said        amino acid sequence, Nanobody or polypeptide of the invention        (also referred to herein as a “nucleic acid of the invention”),        optionally followed by:    -   isolating and/or purifying the amino acid sequence, Nanobody or        polypeptide of the invention thus obtained.    -   In particular, such a method may comprise the steps of:    -   cultivating and/or maintaining a host of the invention under        conditions that are such that said host of the invention        expresses and/or produces at least one amino acid sequence,        Nanobody and/or polypeptide of the invention; optionally        followed by:    -   isolating and/or purifying the amino acid sequence, Nanobody or        polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one embodiment of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring GPCR ???? as a template. These and other techniqueswill be clear to the skilled person, and reference is again made to thestandard handbooks, such as Sambrook et al. and Ausubel et al.,mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting embodiment, a genetic construct of theinvention comprises

-   a) at least one nucleic acid of the invention; operably connected to-   b) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   c) one or more further elements of genetic constructs known per se;

in which the terms “regulatory element”, “promoter”, “terminator” and“operably connected” have their usual meaning in the art (as furtherdescribed herein); and in which said “further elements” present in thegenetic constructs may for example be 3′- or 5′-UTR sequences, leadersequences, selection markers, expression markers/reporter genes, and/orelements that may facilitate or increase (the efficiency of)transformation or integration. These and other suitable elements forsuch genetic constructs will be clear to the skilled person, and may forinstance depend upon the type of construct used, the intended host cellor host organism; the manner in which the nucleotide sequences of theinvention of interest are to be expressed (e.g. via constitutive,transient or inducible expression); and/or the transformation techniqueto be used. For example, regulatory requences, promoters and terminatorsknown per se for the expression and production of antibodies andantibody fragments (including but not limited to (single) domainantibodies and ScFv fragments) may be used in an essentially analogousmanner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promotor). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in the host cells mentionedherein; and in particular promoters for the expression in the bacterialcells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycin or ampicillin), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell. For example, leader sequences known per se for theexpression and production of antibodies and antibody fragments(including but not limited to single domain antibodies and ScFvfragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those that can be used for theexpression in the host cells mentioned herein; and in particular thosethat are suitable for expression in bacterial cells, such as thosementioned herein and/or those used in the Examples below. For some(further) non-limiting examples of the promoters, selection markers,leader sequences, expression markers and further elements that may bepresent/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat.Nos. 7,207,410, 5,693,492 and EP 1 085 089. Other examples will be clearto the skilled person. Reference is also made to the general backgroundart cited above and the further references cited herein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, Nanobody orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, BHK-cells (for example        BHK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;

as well as all other hosts or host cells known per se for the expressionand production of antibodies and antibody fragments (including but notlimited to (single) domain antibodies and ScFv fragments), which will beclear to the skilled person. Reference is also made to the generalbackground art cited hereinabove, as well as to for example WO 94/29457;WO 96/34103; WO 99/42077; Frenken et al., (1998), supra; Riechmann andMuyldermans, (1999), supra; van der Linden, (2000), supra; Thomassen etal., (2002), supra; Joosten et al., (2003), supra; Joosten et al.,(2005), supra; and the further references cited herein.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy). For this purpose, thenucleotide sequences of the invention may be introduced into the cellsor tissues in any suitable way, for example as such (e.g. usingliposomes) or after they have been inserted into a suitable gene therapyvector (for example derived from retroviruses such as adenovirus, orparvoviruses such as adeno-associated virus). As will also be clear tothe skilled person, such gene therapy may be performed in vivo and/or insitu in the body of a patient by administering a nucleic acid of theinvention or a suitable gene therapy vector encoding the same to thepatient or to specific cells or a specific tissue or organ of thepatient; or suitable cells (often taken from the body of the patient tobe treated, such as explanted lymphocytes, bone marrow aspirates ortissue biopsies) may be treated in vitro with a nucleotide sequence ofthe invention and then be suitably (re-)introduced into the body of thepatient. All this can be performed using gene therapy vectors,techniques and delivery systems which are well known to the skilledperson, and for example described in Culver, K. W., “Gene Therapy”,1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.);Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, GeneTher. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.: 811 (1997),289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, NatureMedicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. Nos.5,580,859; 5,589,546; or Schaper, Current Opinion in Biotechnology 7(1996), 635-640. For example, in situ expression of ScFv fragments(Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and of diabodies(Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has been described inthe art.

For expression of the Nanobodies in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan for example also be produced in the milk of transgenic mammals, forexample in the milk of rabbits, cows, goats or sheep (see for exampleU.S. Pat. Nos. 6,741,957, 6,304,489 and 6,849,992 for general techniquesfor introducing transgenes into mammals), in plants or parts of plantsincluding but not limited to their leaves, flowers, fruits, seed, rootsor turbers (for example in tobacco, maize, soybean or alfalfa) or in forexample pupae of the silkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides ofthe invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceuticalexpression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, Nanobody orpolypeptide to be obtained.

Thus, according to one non-limiting embodiment of the invention, theamino acid sequence, Nanobody or polypeptide of the invention isglycosylated. According to another non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is non-glycosylated.

According to one preferred, but non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a bacterial cell, in particular a bacterialcell suitable for large scale pharmaceutical production, such as cellsof the strains mentioned above.

According to another preferred, but non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, Nanobodies and the polypeptides of the invention, the aminoacid sequencesn Nanobodies and polypeptides of the invention can beproduced either intracellullarly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or can be produced extracellularly (e.g. inthe medium in which the host cells are cultured) and then isolated fromthe culture medium and optionally further purified. When eukaryotic hostcells are used, extracellular production is usually preferred since thisconsiderably facilitates the further isolation and downstream processingof the Nanobodies and proteins obtained. Bacterial cells such as thestrains of E. coli mentioned above normally do not secrete proteinsextracellularly, except for a few classes of proteins such as toxins andhemolysin, and secretory production in E. coli refers to thetranslocation of proteins across the inner membrane to the periplasmicspace. Periplasmic production provides several advantages over cytosolicproduction. For example, the N-terminal amino acid sequence of thesecreted product can be identical to the natural gene product aftercleavage of the secretion signal sequence by a specific signalpeptidase. Also, there appears to be much less protease activity in theperiplasm than in the cytoplasm. In addition, protein purification issimpler due to fewer contaminating proteins in the periplasm. Anotheradvantage is that correct disulfide bonds may form because the periplasmprovides a more oxidative environment than the cytoplasm. Proteinsoverexpressed in E. coli are often found in insoluble aggregates,so-called inclusion bodies. These inclusion bodies may be located in thecytosol or in the periplasm; the recovery of biologically activeproteins from these inclusion bodies requires a denaturation/refoldingprocess. Many recombinant proteins, including therapeutic proteins, arerecovered from inclusion bodies. Alternatively, as will be clear to theskilled person, recombinant strains of bacteria that have beengenetically modified so as to secrete a desired protein, and inparticular a amino acid sequence, Nanobody or a polypeptide of theinvention, can be used.

Thus, according to one non-limiting embodiment of the invention, theamino acid sequence, Nanobody or polypeptide of the invention is anamino acid sequence, Nanobody or polypeptide that has been producedintracellularly and that has been isolated from the host cell, and inparticular from a bacterial cell or from an inclusion body in abacterial cell. According to another non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is an amino acid sequence, Nanobody or polypeptide that hasbeen produced extracellularly, and that has been isolated from themedium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left- (PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHOS (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        35S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I);    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1a) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterial cells: pET vectors (Novagen)        and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, SiII, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal;    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (pho1), invertase (Suc), etc.,    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been succesfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence, Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, Nanobody or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or compositions comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one Nanobody of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of theinvention can be formulated and administered in any suitable mannerknown per se, for which reference is for example made to the generalbackground art cited above (and in particular to WO 04/041862, WO04/041863, WO 04/041865 and WO 04/041867) as well as to the standardhandbooks, such as Remington's Pharmaceutical Sciences, 18^(th) Ed.,Mack Publishing Company, USA (1990) or Remington, the Science andPractice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins(2005).

For example, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be formulated and administered in any manner known perse for conventional antibodies and antibody fragments (including ScFv'sand diabodies) and other pharmaceutically active proteins. Suchformulations and methods for preparing the same will be clear to theskilled person, and for example include preparations suitable forparenteral administration (for example intravenous, intraperitoneal,subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be administered using gene therapy methods of delivery. See,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in itsentirety. Using a gene therapy method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, Nanobody orpolypeptide of the invention can additionally be transfected with tissuespecific promoters to target specific organs, tissue, grafts, tumors, orcells and can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of theinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle such as an inert diluent oran assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, Nanobody or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the amino acid sequences, Nanobodies and polypeptides of theinvention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,Nanobodies and polypeptides of the invention may be incorporated intosustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the amino acid sequences, Nanobodies andpolypeptides of the invention or their salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid sequences, Nanobodies and polypeptides of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be applied in pure form, i.e., whenthey are liquids. However, it will generally be desirable to administerthem to the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the amino acid sequences, Nanobodies and polypeptidesof the invention can be dissolved or dispersed at effective levels,optionally with the aid of non-toxic surfactants. Adjuvants such asfragrances and additional antimicrobial agents can be added to optimizethe properties for a given use. The resultant liquid compositions can beapplied from absorbent pads, used to impregnate bandages and otherdressings, or sprayed onto the affected area using pump-type or aerosolsprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the amino acid sequences, Nanobodies and polypeptides of theinvention to the skin are known to the art; for example, see Jacquet etal. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith etal. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptidesof the invention can be determined by comparing their in vitro activity,and in vivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequence, Nanobodies andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides ofthe invention required for use in treatment will vary not only with theparticular amino acid sequence, Nanobody or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the amino acid sequences, Nanobodies and polypeptides of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one IL-6R related disorders, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with IL-6R, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signalling in which IL-6R is involved, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can be treatedby modulating IL-6R, its biological or pharmacological activity, and/orthe biological pathways or signalling in which IL-6R is involved, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, said pharmaceutically effective amount may be an amountthat is sufficient to modulate IL-6R, its biological or pharmacologicalactivity, and/or the biological pathways or signalling in which IL-6R isinvolved

The invention also relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering of an amino acid sequence of theinvention or polypeptide of the invention to a patient, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of an amino acid sequence of the invention, of a polypeptideof the invention, and/or of a pharmaceutical composition comprising thesame.

In another embodiment, the invention relates to a method forimmunotherapy, and in particular for passive immunotherapy, which methodcomprises administering, to a subject suffering from or at risk of thediseases and disorders mentioned herein, a pharmaceutically activeamount of an amino acid sequence of the invention, of an amino acidsequence of the invention, of a polypeptide of the invention, and/or ofa pharmaceutical composition comprising the same.

In the above methods, the amino acid sequences, Nanobodies and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, Nanobodies and/or polypeptides of the inventionand/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorsewell known to the clinician.

The amino acid sequences, Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same are administeredaccording to a regime of treatment that is suitable for preventingand/or treating the disease or disorder to be prevented or treated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease or disorder to beprevented or treated, the severity of the disease to be treated and/orthe severity of the symptoms thereof, the specific amino acid sequences,Nanobody or polypeptide of the invention to be used, the specific routeof administration and farmaceutical formulation or composition to beused, the age, gender, weight, diet, general condition of the patient,and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, Nanobodies and/or polypeptides of theinvention, or of one or more compositions comprising the same, in one ormore pharmaceutically effective amounts or doses. The specific amount(s)or doses to administered can be determined by the clinician, again basedon the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,Nanobody and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences, Nanobodies and polypeptidesof the invention will generally be administered in an amount between 1gram and 0.01 microgram per kg body weight per day, preferably between0.1 gram and 0.1 microgram per kg body weight per day, such as about 1,10, 100 or 1000 microgram per kg body weight per day, eithercontinuously (e.g. by infusion), as a single daily dose or as multipledivided doses during the day. The clinician will generally be able todetermine a suitable daily dose, depending on the factors mentionedherein. It will also be clear that in specific cases, the clinician maychoose to deviate from these amounts, for example on the basis of thefactors cited above and his expert judgment. Generally, some guidance onthe amounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody orpolypeptide of the invention will be used. It is however within thescope of the invention to use two or more amino acid sequences,Nanobodies and/or polypeptides of the invention in combination.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be used in combination with other pharmaceuticallyactive compounds or principles that are or can be used for theprevention and/or treatment of the diseases and disorders cited herein,as a result of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone IL-6R related disorders.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, Nanobody or polypeptide of theinvention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for the prevention and/or treatment ofIL-6R related disorders, and in particular for the prevention andtreatment of one or more of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, Nanobodies or polypeptides of the invention may also besuitably combined with one or more other active principles, such asthose mentioned herein.

Finally, although the use of the Nanobodies of the invention (as definedherein) and of the polypeptides of the invention is much preferred, itwill be clear that on the basis of the description herein, the skilledperson will also be able to design and/or generate, in an analogousmanner, other (single) domain antibodies against the IL-6 receptor, aswell as polypeptides comprising such (single) domain antibodies (inwhich the terms “domain antibody” and “single domain antibody” havetheir usual meaning in the art.

Thus, one further aspect of the invention relates to domain antibodiesor single domain antibodies against the IL-6 receptor, and topolypeptides that comprise at least one such (single) domain antibodyand/or that essentially consist of such a (single) domain antibody.

In particular, such a (single) domain antibody against the IL-6 receptormay comprise 3 CDR's, in which said CDR's are as defined above for theNanobodies of the invention. For example, such (single) domainantibodies may be the single domain antibodies known as “dAb's”, whichare for example as described by Ward et al, supra, but which have CDR'sthat are as defined above for the Nanobodies of the invention. However,as mentioned above, the use of such “dAb's” will usually have severaldisadvantages compared to the use of the corresponding Nanobodies of theinvention. Thus, any (single) domain antibodies against the IL-6receptor according to this aspect of the invention will preferably haveframework regions that provide these (single) domain antibodies againstthe IL-6 receptor with properties that make them substantiallyequivalent to the Nanobodies of the invention.

Thus, in its broadest sense, the invention relates to an amino acidsequence that essentially consists of four framework regions (FR1 toFR4, respectively) and three complimentarity determining regions (CDR1to CDR3, respectively), and that is directed against (as defined herein)the IL-6 receptor. Such an amino acid sequence preferably containsbetween 80 and 200 amino acid residues, such as between 90 and 150 aminoacid residues, such as about 100-130 amino acid residues (althoughsuitable fragments of such an amino acid sequence—i.e. essentially asdescribed herein for the Nanobodies of the invention or equivalentthereto—may also be used), and is preferably such that it forms animmunoglobulin fold or such that, under suitable conditions, it iscapable of forming an immunoglobulin fold (i.e. by suitable folding).The amino acid sequence is preferably chosen from Nanobodies, domainantibodies, single domain antibodies or “dAb's”, and is most preferablya Nanobody as defined herein. The CDR's may be any suitable CDR's (forwhich reference is made to the disclosure herein), but are preferably asdefined herein.

In further aspects, the invention also relates to proteins andpolypeptides that comprise at least one such amino acid sequence; tonucleic acids encoding such amino acid sequences, proteins andpolypeptides; to methods for preparing such amino acid sequences,proteins and polypeptides; to host cells expressing or capable ofexpressing such amino acid sequences, proteins or polypeptides; tocompositions, and in particular to pharmaceutical compositions, thatcomprise such amino acid sequences, proteins, polypeptides, nucleicacids and/or host cells; and to uses of such amino acid sequences,proteins, polypeptides, nucleic acids, host cells and/or compositions,in particular for prophylactic, therapeutic or diagnostic purposes, suchas the prophylactic, therapeutic or diagnostic purposes mentionedherein. All these aspects will be clear to the skilled person based onthe disclosure herein, and may be essentially the same or equivalent tothe embodiments described herein for the Nanobodies of the invention.

Such an amino acid sequence preferably contains between 80 and 200 aminoacid residues, such as between 90 and 150 amino acid residues, such asabout 100-130 amino acid residues, although suitable fragments of suchan amino acid sequence (i.e. essentially as described herein for theNanobodies of the invention or equivalent thereto) may also be used.

Furthermore, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto other “scaffolds”, including but notlimited to human scaffolds or non-immunoglobulin scaffolds. Suitablescaffolds and techniques for such CDR grafting will be clear to theskilled person and are well known in the art, see for example U.S. Pat.No. 7,180,370, WO 01/27160, EP 0 605 522, EP 0 460 167, U.S. Pat. No.7,054,297, Nicaise et al., Protein Science (2004), 13:1882-1891; Ewertet al., Methods, 2004 October; 34(2):184-199; Kettleborough et al.,Protein Eng. 1991 October; 4(7): 773-783; O'Brien and Jones, MethodsMol. Biol. 2003: 207: 81-100; and Skerra, J. Mol. Recognit. 2000: 13:167-187, and Saerens et al., J. Mol. Biol. 2005 Sep. 23; 352(3):597-607,and the further references cited therein. For example, techniques knownper se for grafting mouse or rat CDR's onto human frameworks andscaffolds can be used in an analogous manner to provide chimericproteins comprising one or more of the CDR's of the Nanobodies of theinvention and one or more human framework regions or sequences.

Thus, in another embodiment, the invention comprises a chimericpolypeptide comprising at least one CDR sequence chosen from the groupconsisting of CDR1 sequences, CDR2 sequences and CDR3 sequencesmentioned herein for the Nanobodies of the invention. Preferably, such achimeric polypeptide comprises at least one CDR sequence chosen from thegroup consisting of the CDR3 sequences mentioned herein for theNanobodies of the invention, and optionally also at least one CDRsequence chosen from the group consisting of the CDR1 sequences and CDR2sequences mentioned herein for the Nanobodies of the invention. Forexample, such a chimeric polypeptide may comprise one CDR sequencechosen from the group consisting of the CDR3 sequences mentioned hereinfor the Nanobodies of the invention, one CDR sequence chosen from thegroup consisting of the CDR1 sequences mentioned herein for theNanobodies of the invention and one CDR sequence chosen from the groupconsisting of the CDR1 sequences and CDR2 sequences mentioned herein forthe Nanobodies of the invention. The combinations of CDR's that arementioned herein as being preferred for the Nanobodies of the inventionwill usually also be preferred for these chimeric polypeptides.

In said chimeric polypeptides, the CDR's may be linked to further aminoacid sequences sequences and/or may be linked to each other via aminoacid sequences, in which said amino acid sequences are preferablyframework sequences or are amino acid sequences that act as frameworksequences, or together form a scaffold for presenting the CDR's.Reference is again made to the prior art mentioned in the lastparagraph. According to one preferred embodiment, the amino acidsequences are human framework sequences, for example V_(H)3 frameworksequences. However, non-human, synthetic, semi-synthetic ornon-immunoglobulin framework sequences may also be used. Preferably, theframework sequences used are such that (1) the chimeric polypeptide iscapable of binding the IL-6 receptor, i.e. with an affinity that is atleast 1%, preferably at least 5%, more preferably at least 10%, such asat least 25% and up to 50% or 90% or more of the affinity of thecorresponding Nanobody of the invention; (2) the chimeric polypeptide issuitable for pharmaceutical use; and (3) the chimeric polypeptide ispreferably essentially non-immunogenic under the intended conditions forpharmaceutical use (i.e. indication, mode of administration, dosis andtreatment regimen) thereof (which may be essentially analogous to theconditions described herein for the use of the Nanobodies of theinvention).

According to one non-limiting embodiment, the chimeric polypeptidecomprises at least two CDR sequences (as mentioned above) linked via atleast one framework sequence, in which preferably at least one of thetwo CDR sequences is a CDR3 sequence, with the other CDR sequence beinga CDR1 or CDR2 sequence. According to a preferred, but non-limitingembodiment, the chimeric polypeptide comprises at least two CDRsequences (as mentioned above) linked at least two framework sequences,in which preferably at least one of the three CDR sequences is a CDR3sequence, with the other two CDR sequences being CDR1 or CDR2 sequences,and preferably being one CDR1 sequence and one CDR2 sequence. Accordingto one specifically preferred, but non-limiting embodiment, the chimericpolypeptides have the structure FR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4′, inwhich CDR1, CDR2 and CDR3 are as defined herein for the CDR's of theNanobodies of the invention, and FR1′, FR2′, FR3′ and FR4′ are frameworksequences. FR1′, FR2′, FR3′ and FR4′ may in particular be Framework 1,Framework 2, Framework 3 and Framework 4 sequences, respectively, of ahuman antibody (such as V_(H)3 sequences) and/or parts or fragments ofsuch Framework sequences. It is also possible to use parts or fragmentsof a chimeric polypeptide with the structureFR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4. Preferably, such parts or fragmentsare such that they meet the criteria set out in the preceding paragraph.

The invention also relates to proteins and polypeptides comprisingand/or essentially consisting of such chimeric polypeptides, to nucleicacids encoding such proteins or polypeptides; to methods for preparingsuch proteins and polypeptides; to host cells expressing or capable ofexpressing such proteins or polypeptides; to compositions, and inparticular to pharmaceutical compositions, that comprise such proteinsor polypeptides, nucleic acids or host cells; and to uses of suchproteins or polypeptides, such nucleic acids, such host cells and/orsuch compositions, in particular for prophylactic, therapeutic ordiagnostic purposes, such as the prophylactic, therapeutic or diagnosticpurposes mentioned herein. For example, such proteins, polypeptides,nucleic acids, methods, host cells, compositions and uses may beanalogous to the proteins, polypeptides, nucleic acids, methods, hostcells, compositions and use described herein for the Nanobodies of theinvention.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be obtained in any manner knownper se, for example from Nanobodies (preferred), V_(H) domains fromconventional antibodies (and in particular from human antibodies), heavychain antibodies, conventional 4-chain antibodies (such as conventionalhuman 4-chain antibodies) or other immunoglobulin sequences directedagainst the IL-6 receptor. Such immunoglobulin sequences directedagainst the IL-6 receptor can be generated in any manner known per se,as will be clear to the skilled person, i.e. by immunization with theIL-6 receptor or by screening a suitable library of immunoglobulinsequences with the IL-6 receptor, or any suitable combination thereof.Optionally, this may be followed by techniques such as random orsite-directed mutagenesis and/or other techniques for affinitymaturation known per se. Suitable techniques for generating suchimmunoglobulin sequences will be clear to the skilled person, and forexample include the screening techniques reviewed by Hoogenboom, NatureBiotechnology, 23, 9, 1105-1116 (2005). Other techniques for generatingimmunoglobulins against a specified target include for example theNanoclone® technology (as for example described in the published USpatent application 2006-0211088), so-called SLAM technology (as forexample described in the European patent application 0 542 810), the useof transgenic mice expressing human immunoglobulins or the well-knownhybridoma techniques (see for example Larrick et al, Biotechnology, Vol.7, 1989, p. 934). All these techniques can be used to generateimmunoglobulins against the IL-6 receptor, and the CDR's of suchimmunoglobulins can be used in the Nanobodies of the invention, i.e. asoutlined above. For example, the sequence of such a CDR can bedetermined, synthesized and/or isolated, and inserted into the sequenceof a Nanobody of the invention (e.g. so as to replace the correspondingnative CDR), all using techniques known per se such as those describedherein, or Nanobodies of the invention containing such CDR's (or nucleicacids encoding the same) can be synthesized de novo, again using thetechniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, polypeptides,nucleic acids, genetic constructs and hosts and host cells of theinvention will be clear to the skilled person based on the disclosureherein. For example, and without limitation, the amino acid sequences ofthe invention can be linked to a suitable carrier or solid support so asto provide a medium than can be used in a manner known per se to purifyIL-6R from compositions and preparations comprising the same.Derivatives of the amino acid sequences of the invention that comprise asuitable detectable label can also be used as markers to determine(qualitatively or quantitatively) the presence of IL-6R in a compositionor preparation or as a marker to selectively detect the presence ofIL-6R on the surface of a cell or tissue (for example, in combinationwith suitable cell sorting techniques).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described by means of the followingnon-limiting examples and figures, in which the Figures show:

FIG. 1 Analysis of immune response in llamas 81 and 82 by ELISA.

FIG. 2-FIG. 2E (FIG. 2: Panels A-D; FIG. 2A: Panels E-G; FIG. 2B: PanelsH-K; FIG. 2C: Panels L-O; FIG. 2D: Panels P-S; FIG. 2E: Panels T-W) FACSanalysis of immune response in llamas 081 and 082.

FIG. 3 Schematic representation of Alphascreen assays used to identifyNanobodies against the IL6 binding site on IL6R.

FIG. 4 SDS-PAGE of purified monovalent anti IL6R Nanobodies. M=molecularweight markers. The identity of the Nanobodies is shown on top of thegel.

FIG. 5A-FIG. 5B Protein sequences of anti-IL6R Nanobodies.

FIG. 6 Protein sequences of a selected subset of inhibitory anti-IL6RNanobodies.

FIG. 7-FIG. 7A (FIG. 7: Panels A-D; FIG. 7A: Panels E-H) Binding ofmonovalent inhibitory anti IL6R Nanobodies to IL6-R on U266 cells.Fluorescence intensity is blotted on the X-axis, the number of events onthe Y-axis. Purified anti-IL6-R antibody BR-6 was included.

FIG. 8-FIG. 8A (FIG. 8: Panels A-C; FIG. 8A: Panels D-F), FIG. 9-FIG. 9A(FIG. 9: Panels A-C; FIG. 9A: Panels D-F) and FIG. 10-FIG. 10A (FIG. 10:Panels A-C; FIG. 10A: Panels D-E) Antagonistic activity of 14 monovalentanti IL6R Nanobodies in alphascreen for inhibition of the IL6/IL6-Rinteraction.

FIG. 11-FIG. 11A (FIG. 11: Panels A-H; FIG. 11A: Panels I-P)Antagonistic activity of 14 monovalent anti IL6R Nanobodies incell-based assay (XG-1). BR6, BN12 and the Reference-Fab were includedas a reference.

FIG. 12-FIG. 12A (FIG. 12: Panels A-E; FIG. 12A: Panels F-J):Competitive binding of monovalent anti IL6R Nanobodies to sIL6-R inhuman plasma.

FIG. 13-FIG. 13A (FIG. 13: Panels A-D; FIG. 13A: Panels E-H) Competitivebinding of monovalent anti IL6R Nanobodies to sIL6-R in cynomolgusplasma.

FIG. 14 SDS-PAGE of purified bispecific anti IL6R/anti SA Nanobodies.M=molecular weight markers. The identity of the Nanobodies is shown ontop of the gel.

FIG. 15-FIG. 15A (FIG. 15: Panels A-B; FIG. 15A: Panels C-G) Binding ofbispecific anti IL6R/anti SA Nanobodies to IL6-R on U266 cells.Fluorescence intensity is blotted on the X-axis, the number of events onthe Y-axis. Purified anti-IL6-R antibody BR-6 was included.

FIG. 16-FIG. 16A (FIG. 16: Panels A-C; FIG. 16A: Panels D-F) and FIG.17-FIG. 17A (FIG. 17: Panels A-C; FIG. 17A: Panels D-F) Antagonisticactivity of bispecific anti IL6R/anti SA Nanobodies in Alphascreen forinhibition of the IL6/IL6-R interaction.

FIG. 18-FIG. 18A (FIG. 18: Panels A-C; FIG. 18A: Panels D-F), FIG.19-FIG. 19A (FIG. 19: Panels A-C; FIG. 19A: Panels D-F) and FIG. 20(Panels A-C) Antagonistic activity of bispecific anti IL6R/anti SANanobodies in cell-based assay (XG-1). BR6, BN12 and the Reference-Fabwere included as a reference.

FIG. 21-FIG. 21A (FIG. 21: Panels A-C; FIG. 21A: Panels D-F) and FIG. 22(Panels A-D) Antagonistic activity of bispecific anti IL6R/anti SANanobodies in the presence of 1 mg/mL human serum albumin in cell-basedassay (XG-1). BR6, BN12 and the Reference-Fab were included as areference.

FIG. 23 SDS-PAGE of purified trivalent anti IL6R/anti SA Nanobodies.M=molecular weight markers. The identity of the Nanobodies is shown ontop of the gel.

FIG. 24 Binding of trivalent anti IL6R/anti SA Nanobodies to IL6-R inBiacore.

FIG. 25 (Panels A-D) Binding of trivalent anti IL6R/anti SA Nanobodiesto IL6-R on U266 cells. Fluorescence intensity is blotted on the X-axis,the number of events on the Y-axis. Purified anti-IL6-R antibody BR-6was included.

FIG. 26 (Panels A-F) Antagonistic activity of trivalent anti IL6R/antiSA Nanobodies in the absence and in the presence of 1 mg/mL human serumalbumin in cell-based assay (XG-1).

FIG. 27 Sequences of different humanized variants of IL6R03, IL6R04 andIL6R13.

FIG. 28, FIG. 29, FIG. 30 and FIG. 31 Antagonistic activity of humanizedvariants of IL6R03, IL6R04 and IL6R13 anti IL6R Nanobodies inAlphascreen for inhibition of the IL6/IL6-R interaction.

FIG. 32 and FIG. 33 Concentration of humanized variants of IL6R03,IL6R04 and IL6R13 anti IL6R Nanobodies after incubation at differenttemperatures.

FIG. 34 (Panels A-C) Antagonistic activity in cell-based assay (XG-1) ofhumanized Nanobodies.

FIG. 35 (Panels A-F) Flow cytometric analysis of IMAC-purifiedNanobodies from selected clones. IMAC-purified Nanobodies IL6R-24,IL6R-44, IL6R-202 and IL6R-203 were added to cyno IL6R-positive CHOcells. Detection was performed using a PE-labeled rabbit polyclonalanti-Nanobody serum (R23). Nanobodies binding to cells was measured byan increase in fluorescence intensity as compared to cells that wereincubated with FACS buffer (PBS+10% FBS) followed by PE-labeled R23antiserum. Fluorescence intensity is blotted on the X-axis, the numberof events on the Y-axis.

FIG. 36 and FIG. 37 Binding inhibition curve for ALEXA⁶⁴⁷-labeled hIL-6(625 pM final) competed with unlabeled hIL-6, Nanobody IL6R-202 andNanobody IL6R-203. The average fluorescence and standard deviation isplotted against the concentration of unlabeled competitor. IC₅₀ valueswere calculated in graph pad Prism.

FIG. 38-FIG. 38A (FIG. 38: Panels A-B; FIG. 38A: Panels C-G) Flowcytometric analysis of purified IL6R04-HSA and HSA-IL6R04 fusionproteins and IL6R04. Purified Nanobody constructs were added to U266cells. Detection was performed using a PE-labeled rabbit polyclonalanti-Nanobody serum (R23). Nanobody-HSA fusion protein and Nanobodybinding to cells was measured by an increase in fluorescence intensityas compared to cells that were incubated with FACS buffer (PBS+10% FBS)followed by PE-labeled R23 antiserum. Fluorescence intensity is blottedon the X-axis, the number of events on the Y-axis. Purified anti-IL6Rantibodies BR6 and BN12 were included as positive control.

FIG. 39 Proliferative response of the TF1 cell line to Nanobody-HSAformats. 1.25×10⁴ cells were seeded in triplicate in the presence of 200pg/ml IL-6 and the indicated dilutions of Nanobody-HSA formats orcontrol antibodies BR-6 or BN-12. The cells were pulsed with 0.5 μCi of[³H]thymidine for the last 6 h of a 72-h culture period, after which theincorporated radioactivity was determined.

FIG. 40 PK analysis of Nanobody IL-6R04-HSA in Balb/c mice. The meanhalf-life of Nanobody IL-6R04-HSA is 1.10 days.

FIG. 41 Mean observed (±SD) plasma concentration-time profiles afterintravenous administration of 2.00 mg/kg of (Panels A, B) IL6R04-HSA,(Panels C,D) IL6R202 and (Panels E,F) IL6R203.

FIG. 42 The level of CRP in cynomolgus monkey for the evaluation of invivo efficacy of anti-IL6R Nanobodies.

FIG. 43, FIG. 44 and FIG. 45 Analysis of epitope specificity of a panelof Nanobodies in comparison with the Reference-Fab and Reference IgG(see Reference Example 1 and SEQ ID NOs: 629 to 632) using ELISA.

FIG. 46 Evaluation of specificity of a panel of Nanobodies in comparisonwith the Reference-Fab (see example 29, first paragraph) using Biacore.

FIG. 47, FIG. 48, FIG. 49 and FIG. 50 (Panels A-B) Potency of anti IL6RNanobodies to inhibit the binding of human IL6 to human, rhesus andcynomolgus monkey soluble IL6R present in plasma.

EXAMPLES

Experimental Part

Interleukin-6 (IL6) is a pleiotropic cytokine involved in manyphysiological processes including regulation of inflammation, immuneresponses and hematopoiesis. IL6 exerts its biological activitiesthrough 2 membrane molecules, a ligand binding 80 kDa chain (IL6-R) anda non-ligand-binding signal transducer gp130. Formation of theIL6-IL6-R-gp130 signaling complex occurs sequentially: first IL6 bindsto IL6-R via interaction site I (Kd: ˜10 nM). Next step is binding ofthis complex to gp130 via interaction sites II and III (Kd: 0.8 nM).Interaction sites II and III are composite sites comprising residues ofboth IL6 and IL6-R. IL6 and IL6-R alone have no detectable affinity forgp130. The exact stoichiometry and composition of the IL6-IL6-R-gp130complex is stil under debate. The crystal structure of IL6-IL6-R-gp130complex has been solved (Boulanger (2003) Science 300, 2101-2104) andsuggests a 2:2:2 stoichiometry. Besides the membrane-bound IL6-R, asoluble form (sIL6-R) can be generated by proteolytic cleavage(TACE/ADAM17) or alternative splicing. The complex of IL6 and sIL6-R canalso bind to gp130. Interestingly, this also happens in cells which donot express endogenous IL6-R. Consequently, cells which release thesIL6-R protein render cells which only express gp130 responsive towardsthe cytokine IL6. This mechanism has been termed trans-signaling.

Overexpression of IL6 has been implicated in the pathogenesis of severalclinical disorders including chronic autoimmune diseases, multiplemyeloma, Castleman's disease, post-menopausal osteoporosis and renalcell carcinoma. To date, a number of inhibitors of the IL6 signalingpathway have been developed including CNTO-328 (anti-IL6 MAb, Centocor),Tocilizumab (anti-IL6-R MAb, Chugai/Roche) and C326 (anti-IL6 avimer,Avidia). CNTO-328 and Tocilizumab are currently in clinical trials forMM, RCC, RA, soJIA, CD and SLE. Tocilizumab is available on the Japanesemarket since 2005 for treatment of Castleman's disease (Actemra).

Materials

-   -   Human IL6 was obtained from Diaclone as a recombinant protein        produced in E. coli.    -   Human bio-IL6 was obtained from Diaclone as human IL6        biotinylated by PE (6 biotins/molecule).    -   Human soluble IL6-R was obtained from Peprotech as a recombinant        protein produced in HEK293 cells and from R&D Systems as a        recombinant protein produced in Sf21 cells.    -   MAb BR-6 is a neutralizing anti-IL6-R monoclonal antibody        obtained from Diaclone.    -   MAb BN-12 is a non-neutralizing anti-IL6-R monoclonal antibody        obtained from Diaclone.    -   MAb M182 is a biotinylated anti-IL6-R monoclonal antibody        obtained from BD Biosciences.    -   Llama IgG (h&I) antibody HRP (horse radish peroxidase)        conjugated is a polyclonal antibody against llama IgG raised in        goat obtained from Bethyl Labs.

The Reference Fab and Reference IgG were generated as described in theReference Example below.

Example 1 Immunizations

Two llamas (081 and 082) were immunized with human IL6-R (Peprotech)according to the scheme outlined in Table C-1.

TABLE C-1 Immunization protocol Day Llama 081 Llama 082 Tissuecollection 0 100 μg  100 μg  10 ml pre-immune blood 7 100 μg  100 μg  —14 50 μg 50 μg — 21 50 μg 50 μg — 28 50 μg 50 μg 10 ml immune blood 3550 μg 50 μg — 39 150 ml immune blood PBL1 lymph node bow biopsy 43 150ml immune blood PBL2 52 50 μg 50 μg 59 100 ml immune blood NC1

After completion of the protocol the immune response in each animal wasanalyzed by ELISA. To this end, biotinylated IL6-R (2 μg/ml) wascaptured in a neutravidin coated microtiter plate. Serial dilutions ofserum samples collected at days 0, 28, 39 and 43 were added (startingdilution: 1/500) and bound llama IgG was detected by addition of goatanti-llama IgG HRP labeled. TMB was used as a substrate. Results areshown in FIG. 1.

Immune responses were also analyzed by FACS: serial dilutions (startingdilution: 1/100) of serum samples collected at days 0, 28 and 39 wereincubated with U266B1 cells (human myeloma). Bound llama IgG wasdetected by goat anti-llama IgG FITC labeled. Results are shown in FIG.2.

Example 2 Library Construction

RNA extracted from peripheral blood lymphocytes and lymph node obtainedfrom llama 081 and 082 was used as starting material for RT-PCR toamplify Nanobody encoding gene fragments. These fragments were clonedinto phagemid vector pAX50. Phage was prepared according to standardmethods (see for example the prior art and applications filed by AblynxN. V. cited herein) and stored after filter sterilization at 4° C. forfurther use. The characteristics of the constructed libraries are shownin Table C-2.

TABLE C-2 Size and percentages of inserts of constructed librariesLibrary size % insert Llama 81 6 × 10⁷ 87 Llama 82 5 × 10⁷ 78

Example 3 Selections

Selections were carried out with the above libraries using variousconditions as summarized in Table C-3.

TABLE C-3 Experimental conditions used in different selection strategiesImmobilization/ Concentration/ Method capture Antigen amount ElutionMagnetic Streptavidin bio-IL6-R 0, 1, 10, 100 ng Trypsin beads SolutionStreptavidin bio-IL6-R 0, 0.01, 0.1, 1 nM Trypsin beads Plate BN-12IL6-R 0, 1, 10, 100 nM Trypsin (Peprotech) Plate BN-12 IL6-R 0, 1, 10,100 nM Trypsin (R&D)

Only a single round of selection was performed for all conditions. Eachselection output was analyzed for enrichment factor (# phage present ineluate relative to control), diversity (Hinfl profiling) and percentageof IL6-R positive clones (ELISA). Based on these parameters the bestselections were chosen for further analysis. To this end, the outputfrom each selection was recloned as a pool into the expression vectorpAX51. Colonies were picked and grown in 96 deep well plates (1 mlvolume) and induced by adding IPTG for Nanobody expression. Periplasmicextracts (volume: ˜80 μl) were prepared according to standard methods(see for example the prior art and applications filed by Ablynx N. V.cited herein).

Example 4 Screening

Periplasmic extracts were analyzed first for their ability to inhibitthe IL6-IL6-R interaction. To this end, 2 independent Alphascreen assayswere set up which are depicted schematically in FIG. 3. In assay 1, theperiplasmic extracts were incubated with biotinylated IL6 (3 nM),soluble IL6 receptor (1 nM), streptavidin coated donor beads and MAbBN-12 coated acceptor beads (20 μg/ml). Nanobodies positive in thisassay could either inhibit the IL6-IL6-R interaction or IL6-R-MAb BN-12interaction. To discriminate between these 2 possibilities a secondassay was set up (Assay 2). In this assay the periplasmic extract wereincubated with bio-IL6-R (0.3 nM), streptavidin coated donor beads andMAb BN-12 coated acceptor beads (10 μg/ml). Nanobodies positive in assay1 but negative in assay 2 were considered as IL6-IL6-R inhibitors.Periplasmic extracts were diluted 25-fold in both assays whichcorresponds roughly to a final concentration of 40 nM.

This resulted in two different subclasses of anti-IL6-R Nanobodies; i.e.

a) Nanobodies against IL6-R that were capable of modulating (e.g.partially or fully reducing or preventing) binding of IL6 to IL6-R. Inthe present example, these were obtained in selections where IL6-R wasimmobilized on MAb BN-12 (although other methods of obtaining suchNanobodies will be clear to the skilled person).

b) Nanobodies against IL6-R that were capable of modulating (e.g.partially or fully reducing or preventing) binding of IL6-R to MAbBN-12. In the present example, these were obtained in alternativeselection strategies where IL6-R was not immobilized on MAb BN-12(although other methods of obtaining such Nanobodies will be clear tothe skilled person).

A statistical overview of the screening effort is shown in Table C-4.Nanobodies showing the strongest inhibition were selected for furthercharacterization.

TABLE C-4 Screening for Nanobodies that inhibit the IL6/IL6-Rinteraction # clones # clones # unique Assay screened # inhibitors (%)sequenced sequences IL6-IL6-R 1536 72 (4.7%) 46 14

Example 5 Nanobody Expression and Purification

Selected Nanobodies were expressed in E. coli as c-myc, His6-taggedproteins in a culture volume of 50 mL. Expression was induced byaddition of 1 mM IPTG and allowed to continue for 4 h at 37° C. Afterspinning the cell cultures, periplasmic extracts were prepared byfreeze-thawing the pellets. These extracts were used as startingmaterial for immobilized metal affinity chromatography (IMAC).Nanobodies were eluted from the column with 150 mM imidazole andsubsequently dialyzed against PBS. Total yield and yield per liter ofcell culture are listed in Table C-5.

SDS-PAGE of purified Nanobodies is shown in FIG. 4.

TABLE C-5 Expression yields of anti-IL6-R Nanobodies in E. coli Nanobodyyield yield ID (mg) yield (mg/l) Nanobody ID yield (mg) (mg/l) PMP40H50.14 0.6 PMP34G9 0.09 1.8 PMP35E11 0.65 2.6 PMP31A4 1.06 4.2 PMP32C90.33 6.5 PMP32E2 1.57 6.3 PMP35H4 0.49 9.8 PMP33A3 0.33 1.3 PMP32E100.78 3.1 PMP34A12 0.57 2.3 PMP30C11 0.63 2.5 PMP28E11 0.08 1.6 PMP35C100.53 2.1 PMP35F4 0.24 1.0

Example 6 Characterization of Monovalent Nanobodies

For simplicity, monovalent clones were renamed. An overview is given inTable C-6 below.

TABLE C-6 Overview of nomenclature of monovalent anti-IL6R Nanobodies IDOriginal name IL6R01 PMP28E11 IL6R02 PMP30C11 IL6R03 PMP31A4 IL6R04PMP32C9 IL6R05 PMP32E10 IL6R06 PMP32E2 IL6R07 PMP33A3 IL6R08 PMP34A12IL6R09 PMP34G9 IL6R10 PMP35C10 IL6R11 PMP35E11 IL6R12 PMP35F4 IL6R13PMP35H4 IL6R14 PMP40H5a) Binding to IL6-R in Biacore

Nanobodies showing the strongest inhibition were selected for off-rateanalysis on Biacore and DNA sequencing (FIG. 5 and Table C-7).

A subset of 14 inhibitory Nanobodies and 3 control Nanobodies (which donot inhibit the interaction between IL6 and IL6-R) were selected forfurther analysis in cell based assays. FIG. 6 shows the proteinsequences of this selected subset of Nanobodies.

Affinity constants (Kd) of these 14 individual inhibitory Nanobodieswere determined by surface plasmon resonance on a Biacore 3000instrument. In brief, IL6-R is amine-coupled to a CM5 sensor chip at adensity of 800-1000 RU. Remaining reactive groups are inactivated.Nanobody binding is assessed at various concentrations ranging from 0.5to 50 nM. Each sample is injected for 4 min at a flow rate of 45 μl/minto allow for binding to chip-bound antigen. Next, binding buffer withoutNanobody is sent over the chip at the same flow rate to allow fordissociation of bound Nanobody. After 10 min, remaining bound analyte isremoved by injecting regeneration solution (Glycine/HCl pH1.5). Bindingcurves obtained at different concentrations of Nanobody are used tocalculate Kd values. In Table C-8, an overview of k_(d)/k_(off), k_(a),and K_(d) values for the selected subset of 14 Nanobodies is shown.

TABLE C-7 Off rates (obtained from Biacore-analysis) of anti-IL6-RNanobodies Clone ID k_(off) (s⁻¹) clone ID k_(off) (s⁻¹) PMP 35 A31.18E−03 PMP 33 B11 1.42E−03 PMP 35 C6 9.32E−04 PMP 33 D1 9.34E−04 PMP35 D10 3.26E−04 PMP 33 H10 6.69E−04 PMP 35 G9 1.86E−03 PMP 33 H71.49E−03 PMP 40 F12 4.39E−04 PMP 35 C10 5.09E−04 PMP 31 C5 1.11E−04 PMP35 E2 7.54E−04 PMP 31 D2 2.81E−04 PMP 35 E11 4.17E−04 PMP 32 C9 1.50E−04PMP 35 G11 2.05E−04 PMP 32 F10 5.80E−04 PMP 30 A10 2.68E−03 PMP 28 A21.24E−03 PMP 33 C10 3.08E−03 PMP 28 C7 1.31E−03 PMP 35 H4 1.78E−04 PMP28 D4 1.85E−03 PMP 30 D4 3.24E−04 PMP 28 F7 1.26E−03 PMP 30 H1 2.83E−04PMP 28 H6 2.47E−03 PMP 33 A2 5.00E−04 PMP 31 B11 1.24E−03 PMP 34 F81.42E−04 PMP 31 B4 1.28E−03 PMP 35 B4 3.03E−04 PMP 31 C8 1.25E−03 PMP 40C9 1/3.6E−04  PMP 31 F4 1.23E−03 PMP 40 H5 1.14E−04 PMP 32 E10 1.27E−03PMP 30 B9 1.56E−04 PMP 32 H5 1.28E−03 PMP 30 F1 1.01E−03 PMP 32 D122.70E−03 PMP 34 B4 9.53E−04 PMP 30 A2 2.57E−03 PMP 34 F10 1.63E−03 PMP30 B6 8.42E−04 PMP 40 A2 5.69E−04 PMP 30 G11 1.64E−03 PMP 28 G3 1.93E−03PMP 34 A12 To be determined PMP 30 C11 2.94E−03 PMP 34 C3 4.35E−04 PMP31 A4 1.60E−03 PMP 35 H7 1.48E−03 PMP 34 C11 3.67E−03 PMP 33 G3 1.19E−03PMP 34 E10 2.00E−03 PMP 34 A5 1.68E−03 PMP 34 G9 1.39E−03 PMP 34 D23.31E−04 PMP 35 F4 8.96E−04 PMP 34 E9 5.03E−04 PMP 28 B1 1.34E−03 PMP 34G3 1.40E−04 PMP 28 E11 To be determined PMP 30 B1 1.00E−03 PMP 32 E28.86E−04 PMP 28 G1 3.17E−03 PMP 33 A3 2.42E−04 PMP 30 B3 2.96E−04 PMP 28B2 5.39E−03 PMP 30 B7 6.66E−04 PMP 28 D1 1.45E−02

TABLE C-8 Overview of k_(d)/k_(off)-, k_(a)-, and K_(d)-values for aselected subset of 14 inhibitory anti-IL6-R Nanobodies. Nanobody IDk_(d)/k_(off) (s⁻¹) k_(a) (1/Ms) K_(d) (nM) IL6R01 1.10E−04 2.62E+050.418 IL6R02 2.94E−03 8.40E+05 5.90 4.95E−03 IL6R03 1.47E−03 4.84E+053.03 1.60E−03 IL6R04 9.42E−05 3.65E+05 0.26 1.50E−04 IL6R05 1.41E−031.44E+05 9.79 1.27E−03 IL6R06 8.86E−04 1.07E+06 7.10 7.57E−03 IL6R072.42E−04 IL6R08 1.97E−03 1.94E+05 10.2 IL6R09 1.29E−03 6.41E+05 2.011.30E−03 1.11E+06 1.17 1.39E−03 IL6R10 5.26E−04 4.14E+05 1.27 5.09E−04IL6R11 3.40E−04 3.91E+05 0.87 3.96E−04 2.15E+05 1.85 4.17E−04 IL6R121.16E−03 6.78E+05 1.71 8.96E−04 IL6R13 1.21E−04 2.31E+05 0.53 1.09E−041.37E+05 0.79 1.78E−04 IL6R14 1.00E−04 4.02E+05 0.25 1.14E−04 ReferenceFab 6.60E−04 7.68E+05 0.86b) Binding to IL6-R on U266 Cells

Binding to membrane-bound IL6R expressed on U266 cells was analyzed inFACS. Flow cytometric analysis was performed of IMAC-purified Nanobodiesfrom selected clones (IL6R04, IL6R09, IL6R11, IL6R13, IL6R14).IMAC-purified Nanobodies were added to IL6-R positive U266 cells.Detection was performed by a monoclonal anti-myc antibody followed by aPE-labeled polyclonal anti-mouse antibody (Jackson ImmunoResearchLaboratories 115-115-164 Lot 69854). Nanobodies binding to cells wasmeasured by an increase in fluorescence intensity as compared to cellsthat were incubated with FACS buffer (PBS+10% FBS) followed bymonoclonal anti-myc antibody and/or PE-labeled polyclonal anti-mouseantibody. Results are shown in FIG. 7. Fluorescence intensity is blottedon the X-axis, the number of events on the Y-axis. Purified anti-IL6-Rantibody BR-6 was included.

c) Epitope Mapping

Nanobodies were analyzed for competition with Tocilizumab-Fab. The 14purified Nanobodies were tested in Alphascreen for inhibition of theReference-Fab/IL6R interaction. A fixed concentration of purifiedproteins (100 nM) was added to biotinylated IL6-R (1 nM) and incubatedfor 15 min. Subsequently Reference-Fab-coated acceptor beads were addedand this mixture was incubated for 1 hour. Finally streptavidin donorbeads were added and after 1 hour incubator the plate was read on theEnvision microplate reader. BR-6, BN12 and the Reference-Fab wereincluded as reference. Results, shown in Table C-9, are expressed as the% binding retained in competition with Reference-Fab. The lower thenumber, the higher the competition, the higher the overlap in epitope.

TABLE C-9 Inhibition of the Reference-Fab/IL6R interaction by 14selected inhibitory anti-IL6-R Nanobodies. % binding retained inNanobody ID competition with Reference-Fab IL6R01 49 IL6R02 86 IL6R03 5IL6R04 50 IL6R05 64 IL6R06 36 IL6R07 80 IL6R08 99 IL6R09 62 IL6R10 102IL6R11 40 IL6R12 103 IL6R13 25 IL6R14 96d) Competition Assays/Antagonistic Activity in Alphascreen

The 14 purified Nanobodies were tested in Alphascreen for inhibition ofthe IL6/IL6-R interaction. Serial dilutions of purified proteins(concentration range: 500 nM-10 pM) were added to IL6-R (0.3 nM) andincubated for 15 min. Subsequently 3 nM bio-IL6 and BN12-coated acceptorbeads were added and this mixture was incubated for 1 hour. Finallystreptavidin donor beads were added and after 1 hour incubator the platewas read on the Envision microplate reader. BR-6 and the Reference-Fabwere included as reference. Results are shown in FIGS. 8, 9 and 10.Dose-response curves were observed for all 14 Nanobodies withIC₅₀-values ranging from 48 pM to 1.7 nM. As can be seen from FIGS. 8, 9and 10, the invention provides Nanobodies that, under the conditions ofthis assay, exhibit essentially full (i.e. >90%; preferably >95%)inhibition of the IL6-IL6-R interaction; or alternatively Nanobodiesthat, under the conditions of the assay, provide partial inhibition(i.e. between 25% and 90%; preferably between 40% and 75%) of theIL6-IL6-R interaction (e.g. IL6-R07).

e) Antagonistic Activity in Cell-Based Assay (XG-1)

All purified Nanobodies were tested in the XG1 assay. XG1 is anIL6-dependent human myeloma cell line. Half-maximal proliferation isachieved at ˜20 pg/ml of IL6. Assays were essentially performed asdescribed by Zhang et al. (Blood 83: 3654-3663). BR6, BN12 and theReference-Fab were included as a reference. Results are outlined in FIG.11. IC50 values ranged from 50 nM to 90 pM and are presented in TableC-10.

TABLE C-10 IC50 values of 14 selected inhibitory anti-IL6-R Nanobodiesmeasured in XG-1 cell based assay ID IC50 (nM) IL6R02 31.04 IL6R03 16.16IL6R04 0.089 IL6R05 7.295 IL6R06 42.05 IL6R07 50.47 IL6R08 36.6 IL6R092.745 IL6R10 2.546 IL6R11 5.366 IL6R12 2.753 IL6R13 1.395 IL6R14 0.603Reference-Fab 5.985 BN12 0.2721 BR6 0.064f) Potency of Monovalent Wild Type Nanobodies in Cell-Based Assay (TF-1)

The TF-1 cell (ECACC) line was maintained between 2-9×100,000 cells/mLusing RPMI 1640 supplemented with 2 mM Glutamine, 1% Sodium pyruvate, 3ng/mL Human GM-CSF (eBiosciences) and 10% Foetal Bovine serum (Gibco).Cells were subcultured 3 times a week and were maintained at 37% and a5% CO₂ atmosphere. The same batch of GM-CSF (Lot E019991) and of FoetalBovine Serum (lot no 41Q4556K) was used.

The cell-based assay was performed similarly as described in de Hon, F.D., Ehlers, M., Rose-John, S., Ebeling, S. B., Bos, H. K., Aarden, L.A., and Brakenhoff, J. P. (1994) J Exp Med 180, 2395-2400. Cellsuspensions were centrifuged for 5 min at 200 g and the supernatant wasremoved. Cells were resuspended in RPMI 1640 supplemented with 2 mMGlutamine, 1% Sodium pyruvate and 10% Foetal Bovine serum, were seededat a density of 12500 cells/well in a 96-well plate and incubated for 72h with different dilutions of Nanobodies® and a constant amount of 500pg/mL IL-6. The 96-well plates were incubated in a humid chamber. Everysample was analysed in triplicate. The total volume/well was 200 μL.During the last 6 h of the incubation, cells were pulse-labeled with 0.2μCi/well of ³H-thymidine (GE Healthcare) in a total volume of 20 μL.Cells were harvested with a semiautomatic cell harvester (Filtermateharvester, PerkinElmer) and the ³H-thymidine incorporation was measuredusing a Topcount NXT counter (PerkinElmer). Results are expressed asaverage counts per minute (cpm) per well. IC 50 values are summarised inTable C-11.

TABLE C-11 IC50 values obtained in TF-1 assay of monovalent wild typeNanobodies IC50 (nM) TF-1 IL6R04 0.545 IL6R14 1.751 IL6R11 6.623 IL6R126.915 IL6R13 7.197 IL6R10 9.147 IL6R09 10.01 IL6R05 19.94 IL6R03 28.05IL6R02 29.66 IL6R07 35 IL6R08 42.88 IL6R06 77.23g) Binding to sIL6-R in Human Plasma

Soluble human IL6-R is present in plasma in the range of 80 ng/ml-400ng/ml (Jones et al., 2001, FASEB Journal 15:43-58). To analyze whetherNanobodies bind to naturally occurring sIL6R, binding of monovalentnanobodies to U266 cells was performed in presence of human plasma.Competitive binding could be demonstrated indicating that soluble IL6-Ris bound by the Nanobodies analyzed. Inhibition of binding of monovalentIMAC-purified Nanobodies from selected clones (IL6R03, IL6R04, IL6R13)to U266 cells in the presence of human plasma was measured.IMAC-purified Nanobodies were added to IL6-R positive U266 cells in thepresence of human plasma. Detection was performed by a monoclonalanti-myc antibody followed by a PE-labeled polyclonal anti-mouseantibody. Inhibition of binding of Nanobodies to cells in the presenceof human plasma was measured by a decrease in fluorescence intensity ascompared to the fluorescence intensity of Nanobodies binding to cells inthe absence of human plasma. Results are shown in FIG. 12. Fluorescenceintensity is blotted on the X-axis, the number of events on the Y-axis.Purified anti-IL6-R antibody BR-6 was included. U266 cells incubatedwith monoclonal anti-myc antibody followed by a PE-labeled polyclonalanti-mouse antibody in the presence/absence of human plasma served asbackground staining control.

h) Binding to sIL6-R in Cynomolgus Plasma

Soluble human IL6-R is present in plasma in the range of 80 ng/ml-400ng/ml (Jones et al., 2001, FASEB Journal 15:43-58). To analyze whetherNanobodies bind to naturally occurring sIL6R, binding of monovalentnanobodies to U266 cells was performed in presence of cynomolgus plasma.Competitive binding could be demonstrated indicating that soluble IL6-Ris bound by the Nanobodies analyzed. Inhibition of binding of monovalentIMAC-purified Nanobodies from selected clones (IL6R04, IL6R13) to U266cells in the presence of cynomolgus plasma. IMAC-purified Nanobodieswere added to IL6-R positive U266 cells in the presence of cynomolgusplasma was measured. Detection was performed by a monoclonal anti-mycantibody followed by a PE-labeled polyclonal anti-mouse antibody.Inhibition of binding of Nanobodies to cells in the presence ofcynomolgus plasma was measured by a decrease in fluorescence intensityas compared to the fluorescence intensity of Nanobodies binding to cellsin the absence of cynomolgus plasma. Results are shown in FIG. 13.Fluorescence intensity is blotted on the X-axis, the number of events onthe Y-axis. Purified anti-IL6-R antibody BR-6 was included. U266 cellsincubated with monoclonal anti-myc antibody followed by a PE-labeledpolyclonal anti-mouse antibody in the presence/absence of cynomolgusplasma served as background staining control.

i) Cross-Reactivity to Mouse IL6-R

Binding to mouse IL6-R (R&D Systems, cat #1830-SR/CF) was analyzed inELISA. A maxisorp 96-well plate was coated with mouse IL6-R (1 μg/ml),blocked and incubated with a dilution series of Nanobodies (500 nM-0.16nM). Bound Nanobodies were detected using anti-Myc and anti-mouse horseraddish peroxidase using TMB substrate. No binding could be observed.

Example 7 Construction and Expression of Bi-Specific Anti-IL6-RNanobodies

All 14 selected Nanobodies were also expressed as bispecifics consistingof a C-terminal anti-HSA Nanobody (ALB-1), a 9 amino acid Gly/Ser linkerand an N-terminal anti-IL6-R Nanobody. These constructs were expressedin E. coli as c-myc, His6-tagged proteins and subsequently purified fromthe culture medium by immobilized metal affinity chromatography (IMAC)and size exclusion chromotagraphy (SEC). Total yield and yield per literof cell culture are listed in Table C-12. SDS-PAGE of purifiedNanobodies is shown in FIG. 14.

TABLE C-12 Expression yields of bi-specific anti-IL6-R Nanobodies in E.coli Nanobody ID yield (mg) yield (mg/L) PMP28E11-9GS -ALB-1 tbdPMP30C11-9GS -ALB-1 1.06 4.22 PMP31A4-9GS -ALB-1 tbd PMP32C9-9GS -ALB-10.50 1.98 PMP32E2-9GS -ALB-1 1.05 4.19 PMP32E10-9GS -ALB-1 1.25 5.00PMP33A3-9GS -ALB-1 tbd PMP34A12-9GS -ALB-1 2.11 8.44 PMP34G9-9GS -ALB-13.30 13.2 PMP35C10-9GS -ALB-1 0.93 3.74 PMP35E11-9GS -ALB-1 1.82 7.28PMP35F4-9GS -ALB-1 tbd PMP35H4-9GS -ALB-1 1.88 7.52 PMP40H5-9GS-ALB-1tbd

Example 8 Characterization of Bi-Specific Anti-IL6-R Nanobodies

For simplicity, bispecific clones were renamed. An overview is given inTable C-13 below.

TABLE C-13 Overview of nomenclature of bispecific anti-IL6R NanobodiesID Formatted Nanobody IL6R21 Bispecific PMP28E11-9AA GlySer-ALB-1 IL6R22Bispecific PMP30C11-9AA GlySer- ALB-1 IL6R23 Bispecific PMP31A4-9AAGlySer- ALB-1 IL6R24 Bispecific PMP32C9-9AA GlySer- ALB-1 IL6R25Bispecific PMP32E10-9AA GlySer- ALB-1 IL6R26 Bispecific PMP32E2-9AAGlySer- ALB-1 IL6R27 Bispecific PMP33A3-9AA GlySer- ALB-1 IL6R28Bispecific PMP34A12-9AA GlySer- ALB-1 IL6R29 Bispecific PMP34G9-9AAGlySer- ALB-1 IL6R30 Bispecific PMP35C10-9AA GlySer- ALB-1 IL6R31Bispecific PMP35E11-9AA GlySer- ALB-1 IL6R32 Bispecific PMP35F4-9AAGlySer- ALB-1 IL6R33 Bispecific PMP35H4-9AA GlySer- ALB-1 IL6R34Bispecific PMP40H5-9AA GlySer- ALB-1a) Binding to IL6-R in Biacore

Affinity constants (Kd) of the 14 bispecific (anti-IL6R/anti-HSA)Nanobodies (table C-14) were determined by surface plasmon resonance ona Biacore 3000 instrument. In brief, IL6-R is amine-coupled to a CM5sensor chip at a density of 800-1000 RU. Remaining reactive groups areinactivated. Nanobody binding is assessed at various concentrationsranging from 0.5 to 50 nM. Each sample is injected for 4 min at a flowrate of 45 μl/min to allow for binding to chip-bound antigen. Next,binding buffer without Nanobody is sent over the chip at the same flowrate to allow for dissociation of bound Nanobody. After 10 min,remaining bound analyte is removed by injecting regeneration solution(Glycine/HCl pH1.5). Binding curves obtained at different concentrationsof Nanobody are used to calculate Kd values. In Table C-14 an overviewof k_(d)/k_(off), k_(a), and K_(d) values for the 14 bispecificNanobodies is shown.

TABLE C-14 Overview of k_(d)/k_(off)-, k_(a)-, and K_(d)-values forbinding of 14 bispecific (anti-IL6-R/anti-HSA) Nanobodies to IL6-RNanobody ID k_(d)/k_(off) (s⁻¹) k_(a) (1/Ms) K_(d) (nM) IL6R22 5.65E−033.33E+05 16.9 IL6R23 1.46E−03 3.19E+05 4.59 IL6R24 1.10E−04 3.68E+05 0.3IL6R25 1.21E−03 1.18E+05 10.3 IL6R26 6.90E−03 4.46E+05 15.5 IL6R285.26E−04 2.41E+05 2.18 IL6R29 1.49E−03 7.13E+05 2.1 IL6R30 1.20E−031.60E+05 7.52 IL6R31 3.77E−04 1.62E+05 2.32 IL6R32 1.26E−03 1.01E+06 1.3IL6R33 1.25E−04 1.13E+05 1.11 IL6R34 1.08E−04 2.58E+05 0.417b) Binding to Human and Mouse Serum Albumin in Biacore

Binding of Nanobodies® to serum albumin was characterized by surfaceplasmon resonance in a Biacore 3000 instrument, and an equilibriumconstant, K_(D), was determined. In brief, serum albumin from differentspecies was covalently bound to CM5 sensor chips surface via aminecoupling until an increase of 500 response units was reached. Remainingreactive groups were inactivated. Nanobody® binding was assessed using aseries of different concentrations. Each Nanobody® at each concentrationwas injected for 4 minutes at a flow rate of 45 μl/min to allow forbinding to chip-bound antigen. Next, binding buffer without Nanobody®was sent over the chip at the same flow rate to allow dissociation ofbound Nanobody®. After 15 min, remaining bound analyte was removed byinjecting regeneration solution (50 mM NaOH).

From the sensorgrams obtained for the different concentrations of eachanalyte, K_(D) values were calculated via kinetic data analysis. Resultsare presented in Table C-15 below.

c) Binding to IL6-R on U266 Cells

Flow cytometric analysis of bispecific IMAC-purified Nanobodies fromselected clones (IL6R23, IL6R24, IL6R29, IL6R33) was done. IMAC-purifiedNanobodies were added to IL6-R positive U266 cells. Detection wasperformed by a monoclonal anti-myc antibody followed by a PE-labeledpolyclonal anti-mouse antibody. Nanobodies binding to cells was measuredby an increase in fluorescence intensity as compared to cells that wereincubated with FACS buffer (PBS+10% FBS) followed by monoclonal anti-mycantibody and/or PE-labeled polyclonal anti-mouse antibody. Results areshown in FIG. 15. Fluorescence intensity is blotted on the X-axis, thenumber of events on the Y-axis. Purified anti-IL6-R antibody BR-6 wasincluded.

TABLE C-15 Overview of k_(d)/k_(off)-, k_(a)-, and K_(d)-values forbinding of 14 bispecific (anti-IL6-R/anti-HSA) Nanobodies ® to human,mouse, Nano- Human SA Mouse SA Cyno SA body k_(d)/k_(off) k_(a) K_(d)k_(d)/k_(off) k_(a) K_(d) k_(d)/k_(off) k_(a) K_(d) ID (s⁻¹) (1/Ms) (nM)(s⁻¹) (1/Ms) (nM) (s⁻¹) (1/Ms) (nM) IL6R22 3.27E−03 2.94E+05 11.15.08E−02 4.72E+05 108 IL6R23 7.05E−03 4.58E+05 15.4 5.50E−02 2.00E+05275 2.77E−03 1.00E+05 27.6 3.08E−03 1.92E+05 16 IL6R24 3.31E−03 2.21E+0515 4.95E−02 4.06E+05 122 3.34E−03 1.18E+05 28.3 IL6R25 3.17E−03 2.29E+0513.9 4.74E−02 3.89E+05 122 IL6R26 3.13E−03 3.33E+05 9.39 4.38E−026.01E+05 73 IL6R28 3.78E−03 3.56E+05 10.6 4.78E−02 2.65E+05 180 IL6R293.13E−03 2.91E+05 10.8 4.58E−02 5.52E+05 83 3.07E−03 1.62E+05 19 IL6R303.27E−03 2.71E+05 12.1 5.10E−02 4.53E+05 113 IL6R31 3.04E−03 2.27E+0513.4 4.23E−02 4.88E+05 86.8 IL6R32 3.20E−03 3.15E+05 10 5.00E−022.78E+05 179 IL6R33 3.69E−03 1.35E+05 27.3 5.01E−02 5.08E+05 98.64.04E−03 1.65E+05 24.5 IL6R34 4.72E−03 5.15E+05 9.18 4.40E−02 3.97E+05111 3.04E−03 2.03E+05 15 ALB-1 7.16E−04 1.24E+06 0.58 7.25E−03 1.11E+066.5 Nano- Rhesus SA Baboon SA body k_(d)/k_(off) k_(a) K_(d)k_(d)/k_(off) k_(a) K_(d) ID (s⁻¹) (1/Ms) (nM) (s⁻¹) (1/Ms) (nM) IL6R22IL6R23 2.67E−03 1.12E+05 23.8 7.47E−03 3.22E+05 23.2 IL6R24 3.53E−031.25E+05 28.3 4.98E−03 1.24E+05 40.3 IL6R25 IL6R26 IL6R28 IL6R293.29E−03 1.60E+05 20.6 4.53E−03 1.69E+05 26.8 IL6R30 IL6R31 IL6R32IL6R33 3.83E−03 1.56E+05 24.6 6.26E−03 1.94E+05 32.3 IL6R34 3.17E−032.15E+05 14.7 4.31E−03 2.28E+05 18.9 ALB-1 cyno, rhesus and baboon serumalbumind) Epitope Mapping

Bispecific Nanobodies were analyzed for competition with Reference-Fab.The 14 purified bispecific Nanobodies were tested in Alphascreen forinhibition of the Reference-Fab/IL6R interaction. A fixed concentrationof purified proteins (100 nM) was added to biotinylated IL6-R (1 nM) andincubated for 15 min. Subsequently Reference-Fab-coated acceptor beadswere added and this mixture was incubated for 1 hour. Finallystreptavidin donor beads were added and after 1 hour incubator the platewas read on the Envision microplate reader. BR-6, BN12 and theReference-Fab were included as reference. Results, shown in Table C-16,are expressed as the % binding retained in competition withReference-Fab. The lower the number, the higher the competition, thehigher the overlap in epitope.

TABLE C-16 Inhibition of the Reference-Fab/IL6R interaction by 14bispecific (anti-IL6-R/anti-HSA) Nanobodies % binding retained incompetition Nanobody ID with Reference-Fab IL6R22 71 IL6R23 4 IL6R24 35IL6R25 44 IL6R26 25 IL6R28 82 IL6R29 37 IL6R30 78 IL6R31 37 IL6R32 58IL6R33 17 IL6R34 31e) Antagonistic Activity in Alphascreen

The 14 purified bispecific Nanobodies were tested in Alphascreen forinhibition of the IL6/IL6R interaction. Serial dilutions of purifiedproteins (concentration range: 500 nM-10 pM) were added to IL6-R (0.3nM) and incubated for 15 min. Subsequently 3 nM bio-IL6 and BN12-coatedacceptor beads were added and this mixture was incubated for 1 hour.Finally streptavidin donor beads were added and after 1 hour incubatorthe plate was read on the Envision microplate reader. BR-6 and theReference-Fab fragment were included as reference. Dose-response curves,shown in FIGS. 16 and 17, were observed for Nanobodies with IC₅₀-valuesranging from 55 pM to 1.7 nM (Table C-17).

TABLE C-17 IC50 values of 14 bispecific (anti-IL6-R/anti-HSA) NanobodiesID IC50 (M) IL6R21 IL6R22 5.594E−10 IL6R23 IL6R24 1.451E−10 IL6R256.429E−10 IL6R26 1.666E−09 IL6R27 IL6R28 3.259E−10 IL6R29 1.234E−10IL6R30 3.431E−10 IL6R31 1.309E−10 IL6R32 2.678E−10 IL6R33 1.386E−10IL6R34 1.455E−10f) Antagonistic Activity in Cell-Based Assay (XG-1)

All 14 bispecific Nanobodies were tested in the XG1 assay. XG1 is anIL6-dependent human myeloma cell line. Half-maximal proliferation isachieved at ˜20 pg/ml of IL6. Assays were essentially performed asdescribed by Zhang et al. (Blood 83: 3654-3663). BR6, BN12 and theReference-Fab were included as a reference. Results are outlined inFIGS. 18, 19 and 20. IC50 values ranged from 50 nM to 90 pM and arepresented in Table C-18.

TABLE C-18 IC50 values of 14 bispecific (anti-IL6-R/anti- HSA)Nanobodies measured in XG-1 cell based assay ID IC50 (nM) IL6R21 IL6R2250.17 IL6R23 13.97 IL6R24 0.1542 IL6R25 8.363 IL6R26 65.33 IL6R27 IL6R284.364 IL6R29 3.022 IL6R30 27.16 IL6R31 4.566 IL6R32 1.61 IL6R33 1.574IL6R34 0.339

Nanobodies were also analyzed in the presence of 1 mg/mL human serumalbumin. IC50 values range from 17 nM to 100 pM for monovalentNanobodies and from 37 nM to 670 pM for bispecific Nanobodies. Resultsare shown in FIGS. 21 and 22 and Table C-19.

TABLE C-19 IC50 values of monovalent (anti-IL6R) and bispecific (anti-IL6-R/anti-HSA) Nanobodies measured in XG-1 cell based assay ID IC50(nM) IL6R03 17.54 IL6R04 0.099 IL6R09 2.985 IL6R13 1.321 IL6R14 0.751IL6R23 37.26 IL6R24 0.667 IL6R29 8.691 IL6R33 8.071 IL6R34 1.356

Example 9 Construction, Expression and Purification of TrivalentAnti-IL6-R Nanobodies

Nanobodies were also expressed as trivalent bispecifics consisting of anN-terminal anti-IL6R Nanobody, a C-terminal anti-IL6R Nanobody and ananti-HSA Nanobody (ALB-1) in the middle, connecting the differentbuilding blocks with a 9 amino acid Gly/Ser linker (SEQ ID NO's 478 to492). These constructs were expressed in E. coli as c-myc, His6-taggedproteins and subsequently purified from the culture medium byimmobilized metal affinity chromatography (IMAC) and size exclusionchromotagraphy (SEC). SDS-PAGE of purified Nanobodies is shown in FIG.23.

Example 10 Characterization of Trivalent Anti-IL6-R Nanobodies

a) Binding to IL6R on Biacore

Affinity constants (Kd) of the 3 trivalent Nanobodies were determined bysurface plasmon resonance on a Biacore 3000 instrument. In brief, IL6-Ris amine-coupled to a CM5 sensor chip at a density of 800-1000 RU.Remaining reactive groups are inactivated. Nanobody binding is assessedat various concentrations ranging from 0.5 to 50 nM. Each sample isinjected for 4 min at a flow rate of 45 μl/min to allow for binding tochip-bound antigen. Next, binding buffer without Nanobody is sent overthe chip at the same flow rate to allow for dissociation of boundNanobody. After 10 min, remaining bound analyte is removed by injectingregeneration solution (Glycine/HCl pH1.5). Avid binding could bedemonstrated for the trivalent Nanobody IL6R49 (SEQ ID NO 480) ascompared to the corresponding monovalent Nanobody IL6R09 or thebispecific Nanobody IL6R29 (FIG. 24).

b) Binding to IL6R on U266 Cells

Flow cytometric analysis of bispecific trivalent IMAC-purifiedNanobodies from selected clones (IL6R44 (SEQ ID NO 479), IL6R53 (SEQ IDNO 481)). IMAC-purified Nanobodies were added to IL6-R positive U266cells. Detection was performed by a monoclonal anti-myc antibodyfollowed by a PE-labeled polyclonal anti-mouse antibody. Nanobodiesbinding to cells was measured by an increase in fluorescence intensityas compared to cells that were incubated with FACS buffer (PBS+10% FBS)followed by monoclonal anti-myc antibody and/or PE-labeled polyclonalanti-mouse antibody. Results are shown in FIG. 25. Fluorescenceintensity is blotted on the X-axis, the number of events on the Y-axis.Purified anti-IL6-R antibody BR-6 was included.

c) Antagonistic Activity in Cell Based Assay (XG-1)

Purified trivalent Nanobodies IL6R44, IL6R49 and IL6R53 (SEQ ID NO's479, 480 and 481) were tested in the XG1 assay. XG1 is an IL6-dependenthuman myeloma cell line. Half-maximal proliferation is achieved at ˜20pg/ml of IL6. Assays were essentially performed as described by Zhang etal. (Blood 83: 3654-3663). BR6, BN12 and the Reference-Fab were includedas a reference. Results are outlined in FIG. 26.

Example 11 Binding to Human, Cynomolgus Monkey and Mouse Serum Albuminin Biacore of Wild Type Trivalent Nanobody® Constructs

Binding of Nanobodies® to serum albumin was characterized by surfaceplasmon resonance in a Biacore 3000 instrument, and an equilibriumconstant, K_(D), was determined. In brief, serum albumin from differentspecies was covalently bound to CM5 sensor chips surface via aminecoupling until an increase of 500 response units was reached. Remainingreactive groups were inactivated. Nanobody® binding was assessed using aseries of different concentrations. Each Nanobody® at each concentrationwas injected for 4 minutes at a flow rate of 45 μl/min to allow forbinding to chip-bound antigen. Next, binding buffer without Nanobody®was sent over the chip at the same flow rate to allow dissociation ofbound Nanobody®. After 15 min, remaining bound analyte was removed byinjecting regeneration solution (50 mM NaOH).

From the sensorgrams obtained for the different concentrations of eachanalyte, K_(D) values were calculated via kinetic data analysis. Resultsare presented in Table C-20 below.

TABLE C-20 Kd values of trivalent bispecific Nanobodies to serum albuminfrom different species Nanobody ID Human SA Mouse SA Cyno SA IL6R44 51.4nM 993 nM 43 nM IL6R53 35 nM 497 nM Not determined

Example 12 Humanization

DNA fragments encoding humanized versions of Nanobodies® IL6R03, IL6R04and IL6R13 were assembled from oligonucleotides using a PCR overlapextension method (Stemmer et al., 1995). The sequences of differentvariants are shown in FIG. 27.

a) Antagonistic Activity in Alpha Screen

Humanized clones of IL6R03 (IL6R61, IL6R62, IL6R63, IL6R64 and IL6R65;SEQ ID NOs: 609 to 613), IL6R04 (IL6R71, IL6R72, IL6R73, IL6R74 andIL6R75; SEQ ID NOs: 614 to 618) and IL6R13 (IL6R81, IL6R82, IL6R83,IL6R84 and IL6R88; SEQ ID NOs: 619, 620, 621, 622 and 626, respectively)were tested in Alphascreen for inhibition of the IL6/IL6R interaction.Serial dilutions of purified proteins (concentration range: 500 nM-10pM) were added to IL6-R (0.3 nM) and incubated for 15 min. Subsequently3 nM bio-IL6 and BN12-coated acceptor beads were added and this mixturewas incubated for 1 hour. Finally streptavidin donor beads were addedand after 1 hour incubator the plate was read on the Envision microplatereader. BR-6 and the Reference-Fab fragment were included as reference.Dose-response curves are shown in FIGS. 28, 29, 30 and 31. Nosignificant differences between the final different humanized clones(IL6R65, IL6R75 and IL6R88) and their corresponding non-humanizedversions (IL6R03, IL6R04 and IL6R13, respectively) were observed.

b) Temperature Stability Tests

Temperature stability tests were performed for humanized clones ofIL6R03 (IL6R65), IL6R04 (IL6R75) and IL6R13 (IL6R81, IL6R82, IL6R83 andIL6R84). Samples were diluted at 200 μg/ml and divided in 5*2 aliquotscontaining 60 μl. The different vials were incubated each at a giventemperature ranging from 37° C. to 90° C. (37, 50, 70 and 90° C.) for aperiod of 1 hr. (lid temperature: 105° C.) (control was stored at 4°C.). Thereafter, the samples were hold at 25° C. for 2 hrs (rampingrate: 0.05) and stored over night at 4° C. Precipitates were removed bycentrifugation for 30 min at 14.000 rpm. Supernatant was carefullyremoved and further analysed. OD at 280 nm was measured and theconcentration was calculated based on the extinction coefficients.Results are shown in FIGS. 32 and 33.

c) Binding to IL6-R in Biacore

Humanized clones of IL6R03 (IL6R61, IL6R62, IL6R63, IL6R64 and IL6R65),IL6R04 (IL6R71, IL6R72, IL6R73, IL6R74 and IL6R75) and IL6R13 (IL6R81,IL6R82, IL6R83, IL6R84 and IL6R88) were selected for off-rate analysison Biacore and DNA sequencing (Table C-21 and FIG. 27).

Affinity constants (Kd) of these Nanobodies were determined by surfaceplasmon resonance on a Biacore 3000 instrument. In brief, IL6-R isamine-coupled to a CM5 sensor chip at a density of 800-1000 RU.Remaining reactive groups are inactivated. Nanobody binding was assessedat various concentrations ranging from 0.5 to 50 nM. Each sample wasinjected for 4 min at a flow rate of 45 μl/min to allow for binding tochip-bound antigen. Next, binding buffer without Nanobody was sent overthe chip at the same flow rate to allow for dissociation of boundNanobody. After 10 min, remaining bound analyte was removed by injectingregeneration solution (Glycine/HCl pH1.5). Binding curves obtained atdifferent concentrations of Nanobody were used to calculate Kd values.In Table C-21, an overview of k_(d), k_(a), and K_(d) values for thehumanized Nanobodies is shown.

TABLE C-21 Overview of K_(D), k_(a) and k_(d) values for binding ofhumanized anti-IL- 6 receptor Nanobodies ® to the human Il-6 receptor.IL6R IL6R61 KD (nM) 2   ka (1/Ms) 8.50E+05 kd (1/s) 1.70E−03 IL6R62 KD(nM) 2.22 ka (1/Ms) 9.29E+05 kd (1/s) 2.07E−03 IL6R63 KD (nM) 3.69 ka(1/Ms) 9.90E+05 kd (1/s) 3.65E−03 IL6R64 KD (nM) ka (1/Ms) kd (1/s)1.00E−03 IL6R65 KD (nM) 4   ka (1/Ms) 6.00E+05 kd (1/s) 2.35E−03 IL6R71KD (nM) 0.22 ka (1/Ms) 7.03E+05 kd (1/s) 1.53E−04 IL6R72 KD (nM) 0.33 ka(1/Ms) 5.43E+05 kd (1/s) 1.80E−04 IL6R73 KD (nM) 0.33 ka (1/Ms) 6.98E+05kd (1/s) 2.33E−04 IL6R74 KD (nM) 0.16 ka (1/Ms) 7.67E+05 kd (1/s)1.22E−04 IL6R75 KD (nM) <0.1  ka (1/Ms) 1.00E+06 kd (1/s)  <1E−04 IL6R81KD (nM) 0.4  ka (1/Ms) 3.20E+05 kd (1/s) 1.28E−04 IL6R82 KD (nM) 5.07 ka(1/Ms) 6.19E+05 kd (1/s) 3.14E−03 IL6R83 KD (nM) 0.34 ka (1/Ms) 3.50E+05kd (1/s) 1.20E−04 IL6R84 KD (nM) 5.36 ka (1/Ms) 7.62E+05 kd (1/s)4.09E−03 IL6R85 KD (nM) ka (1/Ms) kd (1/s) 2.06E−03 IL6R86 KD (nM) ka(1/Ms) kd (1/s) 1.70E−03 IL6R87 KD (nM) ka (1/Ms) kd (1/s) 1.05E−04IL6R88 KD (nM) 0.9  ka (1/Ms) 2.30E+05 2.13E−04 kd (1/s) 3.10E−04 IL6R89KD (nM) ka (1/Ms) kd (1/s) 1.90E−04 IL6R90 KD (nM) ka (1/Ms) kd (1/s)1.92E−03

Example 13 Antagonistic Activity in Cell-Based Assay (XG-1) of HumanizedNanobodies

Humanized Nanobodies were analysed in the XG-1 assay. XG-1 is anIL6-dependent human myeloma cell line. Half-maximal proliferation isachieved using ˜20 pg/ml IL6. Assays were performed as described byZhang et al. (Blood 83: 3654-3663). BR6, BN12 were included as areference. Results are shown in FIG. 34.

Example 14 Construction of Bivalent and Trivalent Humanized Nanobodies

Nanobodies were constructed to bivalent (SEQ ID NO's 559 to 602) andtrivalent (SEQ ID NO's 493 to 558) constructs using the humanizedanti-IL6R building blocks and humanized anti-serum albumin buildingblocks. A 9-amino acid GlySer linker was used to link the differentbuilding blocks. These constructs were expressed in E. coli and purifiedusing ProtA followed by size exclusion chromatography (SEC).

Example 15 Binding to IL6-R in Biacore of Humanized Bivalent andTrivalent Nanobodies

Affinity constants (Kd) of the bispecific bivalent and trivalenthumanized Nanobodies were determined by surface plasmon resonance on aBiacore 3000 instrument. In brief, IL6-R was amine-coupled to a CM5sensor chip at a density of 800-1000 RU. Remaining reactive groups wereinactivated. Nanobody binding was assessed at various concentrationsranging from 0.5 to 50 nM. Each sample was injected for 4 min at a flowrate of 45 μl/min to allow for binding to chip-bound antigen. Next,binding buffer without Nanobody was sent over the chip at the same flowrate to allow for dissociation of bound Nanobody. After 10 min,remaining bound analyte was removed by injecting regeneration solution(Glycine/HCl pH1.5). Binding curves obtained at different concentrationsof Nanobody were used to calculate Kd values. In Table C-22 an overviewof k_(d)/k_(off), k_(a), and K_(d) values for the bispecific Nanobodiesis shown.

TABLE C-22 Overview of k_(d)/k_(off), k_(a) and K_(d)-values for bindingof bispecific humanized bivalent and trivalent Nanobodies to IL6-RNanobody ID k_(d)/k_(off) (s⁻¹) k_(a) (1/Ms) K_(d) (nM) IL6R2025.7-4.5E+05 1.4-1.45E−04 0.25-0.32 IL6R203     3.8E+05    ~1.5E−05~0.040

Example 16 Binding to Serum Albumin from Different Species of HumanizedBivalent and Trivalent Nanobodies

Binding of Nanobodies® to serum albumin was characterized by surfaceplasmon resonance in a Biacore 3000 instrument, and an equilibriumconstant, K_(D), was determined. In brief, serum albumin from differentspecies was covalently bound to CM5 sensor chips surface via aminecoupling until an increase of 500 response units was reached. Remainingreactive groups were inactivated. Nanobody® binding was assessed using aseries of different concentrations. Each Nanobody® at each concentrationwas injected for 4 minutes at a flow rate of 45 μl/min to allow forbinding to chip-bound antigen. Next, binding buffer without Nanobody®was sent over the chip at the same flow rate to allow dissociation ofbound Nanobody®. After 15 min, remaining bound analyte was removed byinjecting regeneration solution (50 mM NaOH).

From the sensorgrams obtained for the different concentrations of eachanalyte, K_(D) values were calculated via kinetic data analysis. Resultsare presented in Table C-23 below.

TABLE C-23 Overview of k_(d)/k_(off), k_(a) and K_(d)-values for bindingof humanized bivalent and trivalent Nanobodies to serum albumin fromdifferent species Cyno serum albumin Nanobody ID k_(d)/k_(off) (s⁻¹)k_(a) (1/Ms) K_(d) (nM) IL6R202 1.19E+05 5.20E−03 43.6 IL6R203 8.06E+044.36E−03 54.1 Mouse serum albumin Nanobody ID K_(d) (nM) IL6R202 493IL6R203 1009 Human serum albumin Nanobody ID k_(d)/k_(off) (s⁻¹) k_(a)(1/Ms) K_(d) (nM) IL6R202 1.07E+5  5.56E−03 52.1 IL6R203 6.47E+044.53E−03 70

Example 17 Antagonistic Activity in Cell-Based Assay (TF-1)

The TF-1 cell (ECACC) line was maintained between 2-9×100,000 cells/mLusing RPMI 1640 supplemented with 2 mM Glutamine, 1% Sodium pyruvate, 3ng/mL Human GM-CSF (eBiosciences) and 10% Foetal Bovine serum (Gibco).Cells were subcultured 3 times a week and were maintained at 37% and a5% CO₂ atmosphere. The same batch of GM-CSF (Lot E019991) and of FoetalBovine Serum (lot no 41Q4556K) was used.

The cell-based assay was performed similarly as described in de Hon, F.D., Ehlers, M., Rose-John, S., Ebeling, S. B., Bos, H. K., Aarden, L.A., and Brakenhoff, J. P. (1994) J Exp Med 180, 2395-2400. Cellsuspensions were centrifuged for 5 min at 200 g and the supernatant wasremoved. Cells were resuspended in RPMI 1640 supplemented with 2 mMGlutamine, 1% Sodium pyruvate and 10% Foetal Bovine serum, were seededat a density of 12500 cells/well in a 96-well plate and incubated for 72h with different dilutions of Nanobodies® and a constant amount of 500pg/mL IL-6. The 96-well plates were incubated in a humid chamber. Everysample was analysed in triplicate. The total volume/well was 200 μL.During the last 6 h of the incubation, cells were pulse-labeled with 0.2μCi/well of ³H-thymidine (GE Healthcare) in a total volume of 20 μL.Cells were harvested with a semiautomatic cell harvester (Filtermateharvester, PerkinElmer) and the ³H-thymidine incorporation was measuredusing a Topcount NXT counter (PerkinElmer). Results are expressed asaverage counts per minute (cpm) per well. IC 50 values are summarised inTable C-24.

TABLE C-24 IC50 values obtained in TF-1 assay of humanized bivalent andtrivalent Nanobodies IC50 value (nM) Nanobody IC50 (nM) Stand. Dev.IL6R202 0.180 0.061 IL6R203 0.056 0.019

Example 18 Antagonistic Activity in Cell-Based TF-1 Assay in Presence ofSerum Albumin

To test the potential of the bivalent and trivalent Nanobodies to blockthe IL-6/IL6R interaction, a proliferation assay was performed using theIL-6 sensitive TF1 erythroleukemia cell line. Briefly, serial dilutionsof the Nanobodies (pre-incubated with 1% serum albumin) were added to1.25×10⁴ TF-1 cells in triplicate in the presence of a fixedconcentration of IL-6 (200 pg/ml). After 72 h incubation, cells werepulsed with 0.5 μCi of [³H]thymidine and incubated for an additional 6 hprior to freezing at −80° C. Cells were subsequently thawed and embeddedon glass fiber membranes using a cell harvester (Perkin Elmer LifeSciences, Wellesley, Ma., USA). After several washings with water,filters were air-dried and counted using a γ-counter (Perkin Elmer LifeSciences). The monoclonal antibody BR-6 known to neutralize the IL6/IL6Rinteraction was used as positive control. The non-neutralizingmonoclonal antibody BN-12 was used as negative control. Results aresummarized in the table C-25.

TABLE C-25 IC50 values of inhibition of IL-6/IL6R interaction bybivalent and trivalent anti-IL6R Nanobodies Compound IC50 (w/o HSA) IC50(+1% HSA) IL6R24 0.305 ± 0.073 (n = 4) 0.870 ± 0.326 (n = 4) IL6R440.122 ± 0.132 (n = 4) 0.144 ± 0.052 (n = 4) IL6R202 0.190 ± 0.056 (n =2)  0.6 ± 0.141 (n = 2) IL6R203 ND 0.092 (n = 1) BR6 0.042 ± 0.014 (n =2) 0.088 ± 0.036 (n = 3)

Example 19 Binding of Anti-hIL-6R Nanobodies to Cynomolgus Monkey IL-6RPositive Cells

To verify whether the anti-human IL-6R Nanobodies are able to recognizecynomolgus IL-6R, binding to CHO-K1 cells that are stably transfectedwith cynomolgus IL-6R was assessed by flow cytometry.

Cell binding assays were carried out by initially incubating 200,000cells with purified Nanobodies. After incubation, the cells were washedwith FACS buffer. Cells were subsequently incubated with phycoerythrinlabeled rabbit anti-VHH antiserum. To omit signals arising from deadcells a TOPRO-3 (Invitrogen) staining was carried out. Cells werefinally analyzed on a BD FACSArray Bioanalyzer System (BD Biosciences).

FIG. 35 depicts binding of Nanobody constructs to cyno IL-6R transfectedCHO-K1 cells as measured by flow cytometric analysis. It can be seenthat the constructs IL6R-24, IL6R-44, IL6R-202 and IL6R-203 show clearlydiscernable shifts in fluorescence intensity as compared to thefluorescence intensity for cells incubated only with FACS buffer alonein the absence of any construct but with all appropriate detectionagents as used for the detection of Nanobody constructs.

Example 20 Anti-Human IL-6R Nanobodies Compete with Human IL-6 forBinding to Cynomolgus IL-6R

To evaluate the capacity of expressed Nanobodies to block the binding ofhuman IL-6 to cynomolgus IL-6R, a human IL-6 competitive homogeneouscell-based assay was performed. The FMAT 8200 HTS system (AppliedBiosystems) assay was performed as follows: CHO-K1 cells stablyexpressing cynomolgus IL-6R were detached using 0.25% (vol/vol) trypsinand suspended in RPMI supplemented with 10% fetal calf serum (FCS),glutamine, and penicillin-streptomycin. Aliquots of 60 μl (4×10³ cells)were plated into FMAT system 384-well plates (PE Biosystems, CA) andallowed to adhere for 24 h. After overnight adherence, culturesupernatant was removed by gently tapping the plate. To initiate thecompetitive screen, 20 μl ALEXA⁶⁴⁷-labeled human IL-6 (625 pM finalconcentration) and 20 μl Nanobody or cold h-IL6 dilution were added tothe cell-containing FMAT system 384-well plates (PE Biosystems, CA). Theplates were scanned after 10 hours of incubation. A well was consideredpositive if it had a count of over 50 events. FIGS. 36 and 37 depict theresults of experiments measuring the capacity of anti hIL-6R Nanobodiesto block binding of hIL-6 to cynomolgus IL-6R as measured by competitiveFMAT. It can be seen that the constructs IL6R-202 and IL6R-203 clearlycompete with the binding of hIL-6 to cynomolgus IL-6R. The positivecontrol cold hIL-6 shows, as expected, clearly discernable competitionwith ALEXA⁶⁴⁷-labeled hIL-6.

Example 21 Construction of anti-IL-6R Nanobody—Human Serum AlbuminFusion Proteins

HSA was constructed by gene assembly and cloned into pPICZαA asXhoI/NotI fragment.

For the generation of a NB04-HSA fusion protein, NB04 was amplified byPCR using primer pair:

(SEQ ID NO: 634) 5′-GGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTGAGGTGCAGCTGGTGGAGTCTGGG-3′ and (SEQ ID NO: 635) 5′-TCTCTTCTCCTAGGTCTTTGAATCTGTGGGCGACTTCAGATTTATG AGCATCTGAGGAGACGGTGACCTG-3′.

The resulting PCR product was cloned into PCR4TOPO and used to cloneIL6R04 N-terminally of HSA. Hereto, pPICZαA HSA and TOPO NB04-N weredigested with XhoI and AvrII and ligated to each other.

To connect NB04 to the C-terminus of HSA interspaced with a linker, thefollowing nested-PCR was performed: NB04 was first amplified by PCRusing primer pair:

(SEQ ID NO: 636) 5′-GTGGATCCGGAGGCAGTGGAGGTTCTGGTGGGTCAGGAGGTGAGGTGCAGCTGGTGGAGTCTGGG-3′ and (SEQ ID NO: 637) 5′-TAGAAAGCTGGCGGCCGCTTATTATGAGGAGACGGTGACCTG-3′.

A second PCR was performed on the obtained product to insert a GlySerlinker by use of primer pair

(SEQ ID NO: 638) 5′-TTGGTTGCGGCCAGTCAGGCCGCACTTGGTTTGGGTGGATCCGGAGGCAGTG-3′ and (SEQ ID NO: 639) 5′-TAGAAAGCTGGCGGCCGCTTATTATGAGGAGACGGTGACCTG-3′.

The resulting PCR product was cloned into PCR4TOPO and used to cloneNB04 at the C-terminus of HSA. Hereto, pPICZαA HSA and TOPO NB04-C weredigested with NotI and SfiI and ligated to each other.

Example 22 Binding of IL6R04-HSA and HSA-IL6R04 to IL-6R-Positive CellLines in Flow Cytometric Analysis

The test for binding to the IL-6R positive human multiple myeloma cellline U266 was carried out with purified IL6R04-HSA (SEQ ID NO 604),HSA-IL6R04 (SEQ ID NO 603) and IL6R04 (SEQ ID NO 440) using thecommercially available murine anti-human IL6R antibodies BR6 and BN12 aspositive controls.

Cell binding assays were carried out by initially incubating 200,000cells with 5 nM purified Nanobody-HSA fusion proteins. After incubation,the cells were washed with FACS buffer. Cells were subsequentlyincubated with phycoerythrin labeled rabbit anti-VHH antiserum. To omitsignals arising from dead cells a TOPRO-3 (Invitrogen) staining wascarried out. Cells were finally analyzed on a BD FACSArray BioanalyzerSystem (BD Biosciences).

FIG. 38 depicts binding of the Nanobody-HSA fusion constructs to U266cells as measured by flow cytometric analysis. The positive controls BR6and BN12 show, as expected, a clearly discernable shift in fluorescenceintensity as compared to the respective control (anti-IgG^(PE)). Theconstructs IL6R04-HSA and HSA-IL6R04 tested show binding to theIL6R-positive U266 cells as indicated by the shifts in fluorescenceintensity as compared to the fluorescence intensity for cells incubatedonly with FACS buffer in the absence of any construct but with allappropriate detection agents as used for the detection of the Nanobodyconstructs. No obvious difference in binding to U266 cells was observedbetween IL6R04-HSA, HSA-IL6R04 and the monovalent IL6R04 Nanobody.

Example 23 Screening of Kinetic Off-Rate Constants Via Surface PlasmonResonance (BIAcore)

Affinity constants (Kd) of the anti-IL6R Nanobody-HSA constructs weredetermined by surface plasmon resonance on a Biacore 3000 instrument. Inbrief, IL6-R is amine-coupled to a CM5 sensor chip at a density of800-1000 RU. Remaining reactive groups are inactivated. Nanobody bindingis assessed at various concentrations ranging from 0.5 to 50 nM. Eachsample is injected for 4 min at a flow rate of 45 μl/min to allow forbinding to chip-bound antigen. Next, binding buffer without Nanobody issent over the chip at the same flow rate to allow for dissociation ofbound Nanobody. After 10 min, remaining bound analyte is removed byinjecting regeneration solution (Glycine/HCl pH1.5). Binding curvesobtained at different concentrations of Nanobody are used to calculateKd values. The binding characteristics are summarized in Table C-26.

TABLE C-26 Surface plasmon resonance measurements of the interactionbetween hIL-6R and anti-IL6R Nanobody-HSA constructs Ka (1/Ms) Kd (1/s)KD (nM) IL6R04 5.36E+05 1.12E−04 0.20 HSA-IL6R04 2.52E+05 5.62E−05 0.22IL6R04-HSA 1.89E+05 5.60E−05 0.30

Example 24 Anti-IL6R Nanobody-Albumin Fusion can Block hIL-6 InducedProliferation of the Erythroleukemia Cell Line TF1

To test the potential of the anti-IL6R Nanobody-albumin fusions to blockthe IL-6/IL6R interaction, a proliferation assay was performed using theIL-6 sensitive TF1 erythroleukemia cell line. Briefly, serial dilutionsof the anti-IL6R Nanobody-albumin fusions were added to 1.25×10⁴ TF-1cells in triplicate in the presence of a fixed concentration of IL-6(200 pg/ml). After 72 h incubation, cells were pulsed with 0.5 μCi of[³H]thymidine and incubated for an additional 6 h prior to freezing at−80° C. Cells were subsequently thawed and embedded on glass fibermembranes using a cell harvester (Perkin Elmer Life Sciences, Wellesley,Ma., USA). After several washings with water, filters were air-dried andcounted using a γ-counter (Perkin Elmer Life Sciences). The monoclonalantibody BR-6 known to neutralize the IL6/IL6R interaction was used aspositive control. The non-neutralizing monoclonal antibody BN-12 wasused as negative control.

As shown in FIG. 39, both IL6R04-HSA and HSA-IL6R04 can efficientlyblock the IL-6 dependent proliferation of TF1 cells. As expected,antibody BR-6 efficiently blocked IL-6 dependent proliferation of TF1cells whereas BN-12 had no effect.

Example 25 Generation of Multivalent Nanobody-Albumin Fusion ProteinFormats

To potentially increase the biological effect of Nanobody-albuminmolecules, bivalent fusions are generated whereby 2 Nanobodies are fusedat the N-terminal side of serum albumin. The Nanobodies are fusedhead-to-tail using an amino acid linker peptide consisting of differentlengths.

Here we describe the construction and characterization of bivalentNanobody-albumin fusion proteins consisting of two identical anti-IL6-RNanobodies separated by a 3 Ala, 9 (GS) or 20 (GS) amino acid linkerpeptide. DNA segments encoding Nanobody IL6R-201 were head-to-tailgenetically linked and fused to human serum albumin at its N-terminalside, resulting in constructs IL6R201-HSA (SEQ ID NO 605),IL6R201-AAA-IL6R201-HSA (SEQ ID NO 608), IL6R201-9GS-IL6R201-HSA (SEQ IDNO 606), IL6R201-20GS-IL6R201-HSA (SEQ ID NO 607). All Nanobody-albuminfusion proteins were expressed in Pichia pastoris in shake flasks or ina fermentor. Production levels of >50 mg/l were obtained.

Example 26 Fusion of Nanobody to Albumin Increases its Serum Half-Life

The pharmacokinetic study of a HSA fusion Nanobody was conducted inBalbc mice. The mice are preferably 8 up to 12 weeks old. We tested thepharmacokinetic profile from Nanobody, IL6R04-HSA and NanobodyHSA-IL6R04. The Nanobody was injected in three mice. The mice wereadministrated with 200 μg/200 μl via an intravenous infusion. Bloodsamples were taken at 15′, 1 h, 2 h, 4 h, 8 h, 24 h, day 2, day 3, day4, day 5, day 8 and day 15 after the start of infusion. 1000 μl wholeblood was withdrawn per sampling and the serum was isolated after 1 hrincubation at 37° C. The plasma samples are stored by −20° C.

Serum samples were tested for the determination of plasma levels ofhalf-life extension Nanobodies.

Micro titer plates (Maxisorb) are coated with 0.5 μg/ml sIL-6R(Prepotech) in PBS at 100 μl/well overnight at 4° C. The plates arewashed with PBS containing 0.1% Tween20 and blocked for 2 hours at RTwith PBS containing 1% casein. Plasma samples are then diluted in anon-coated plate and incubated for 30 minutes at room temperature. 100μl of all the dilutions is then transferred to the coated plate andallowed to bind for 1 hour at room temperature. After 1 hour, the plateis washed and 100 ul of 1/2000 dilution of biotinylated goat anti-humanalbumin in PBS containing 1% casein is added per well. After furtherincubation for half an hour at room temperature, wells are washed and100 μl of 1/2000 dilution of streptavidin conjugated with horse radishperoxidase in PBS containing 1% casein. This enzyme catalyzes a chemicalreaction with the substrate slow TMB, which results in a colorimetricchange. After stopping this reaction with HCl, the intensity of thecolor can be measured by a spectrophotometer, which determines theoptical density of the reaction product, using a 450 nm wavelength oflight.

The half-life extension Nanobody concentration in the plasma samples isdetermined towards a standard curve. The concentration determination isdetermined with the sigmoidal dose-response curve with variable slope.

FIG. 40 shows that the albumin fusion prolonges the half-life of amonovalant Nanobody targeting IL6R (IL6R-HSA) and has been determined tobe 1.10 days.

The half-life for HSA-IL6R04 has been determined to be 1.0 days.

As a non-formatted Nanobody is cleared within the first hour, theincrease in half-life by fusing with human albumin is more than 25 fold.

Example 27 Pharmacokinetic Profile in Cynomolgus Monkey

A pharmacokinetic study of IL6R202, IL6R203 and IL6R04-HSA was conductedin cynomolgus monkeys. The Nanobody was administered intravenously bybolus injection (1.0 ml/kg, approximately 30 sec) in the vena cephalicaof the left or right arm to obtain a dose of 2.0 mg/kg. Table C-27summarizes the dosing regimen for all monkeys.

TABLE C-27 Dosing regimen of all animals Dose Animal Volume Dose GroupTest item Route Animal ID (ml/kg) (mg/kg) 1 IL6R04- Iv Cynomolgus 1m 1.02.0 HSA (bolus) Monkey Iv Cynomolgus 2 f 1.0 2.0 (bolus) Monkey 2IL6R202 Iv Cynomolgus 3 m 1.0 2.0 (bolus) Monkey Iv Cynomolgus 4 f 1.02.0 (bolus) Monkey 3 IL6R203 Iv Cynomolgus 5 m 1.0 2.0 (bolus) Monkey IvCynomolgus 6 f 1.0 2.0 (bolus) MonkeyThe anti-IL6R Nanobody concentration in the plasma samples wasdetermined as follows:a) Immunodetection of IL6R04-HSA in Plasma Samples

Maxisorb micro titer plates (Nunc, Article No. 430341) were coatedovernight at 4° C. with 100 μl of a 0.5 μg/ml solution of sIL6R(Peprotech, Article No. 20006R) in DPBS (pH 7.4). After coating, theplates were washed three times (PBS containing 0.1% Tween20) and blockedfor 2 hours at room temperature (RT) with PBS containing 1% casein (250μl/well). Plasma samples and serial dilutions of Nanobody-standards(spiked in 100% pooled blank cynomolgus plasma) were diluted in PBS (ina separate non-coated plate) to obtain the desiredconcentration/dilution in a final sample matrix consisting of 10% pooledcynomolgus plasma in PBS. All pre-dilutions were incubated for 30minutes at RT in the non-coated plate. After the blocking step, plateswere washed three times (PBS containing 0.1% Tween20), and an aliquot ofeach sample dilution (100 μl) was transferred to the coated plates andallowed to bind for 1 hour at RT. After sample incubation, the plateswere washed three times (PBS containing 0.1% Tween20) and incubated with100 μl of 1/2000 dilution (in PBS containing 0.1% casein) of polyclonalgoat anti-human serum albumin (Nordic Immunology, Article No. 5184).After 1 hour at RT, the plates were washed three times (PBS containing0.1% Tween20) and incubated with 100 μl of a 1/2000 dilution (in PBScontaining 1% casein) of streptavidine conjugated with horseradishperoxidase (DaktoCytomation, Article No. P0397). After 30 minutes,plates were washed three times (PBS containing 0.1% Tween20) andincubated with 100 μl of slow TMB (Pierce, Article No. 34024). After 20minutes, the reaction was stopped with 100 μl HCl (1N). The absorbanceof each well was measured at 450 nm (Tecan Sunrise spectrophotometer),and corrected for absorbance at 620 nm. This assay measures free andsIL6R-bound IL6R04-HSA Nanobodies. Concentration in each plasma samplewas determined based on a sigmoidal standard curve with variable slopeof the respective Nanobody. The LLOQ and ULOQ of IL6R04-HSA were 2.00ng/ml and 100 ng/ml, respectively. Each individual plasma sample wasanalyzed in three independent assays and an average plasma concentrationwas calculated for pharmacokinetic data analysis.

b) Immunodetection of IL6R202 and IL6R203 in Plasma Samples

Maxisorb micro titer plates (Nunc, Article No. 430341) were coatedovernight at 4° C. with 100 μl of a 5 μg/ml solution of 12B2-GS9-12B2(B2#1302nr4.3.9) in bicarbonate buffer (50 mM, pH 9.6). After coating,the plates were washed three times with PBS containing 0.1% Tween20 andblocked for 2 hours at room temperature (RT) with PBS containing 1%casein (250 μl/well). Plasma samples and serial dilutions ofNanobody-standards (spiked in 100% pooled blank cynomolgus plasma) werediluted in PBS in a separate non-coated plate (Nunc, Article No. 249944)to obtain the desired concentration/dilution in a final sample matrixconsisting of 10% pooled cynomolgus plasma in PBS. All pre-dilutionswere incubated for 30 minutes at RT in the non-coated plate. After theblocking step, the coated plates were washed three times (PBS containing0.1% Tween20), and an aliquot of each sample dilution (100 μl) wastransferred to the coated plates and allowed to bind for 1 hour at RT.After sample incubation, the plates were washed three times (PBScontaining 0.1% Tween20) and incubated for 1 hour at RT with 100 μl of a100 ng/ml solution of sIL6R in PBS (Peprotech, Article No. 20006R).After 1 hour at RT, the plates were washed three times (PBS containing0.1% Tween20) and incubated with 100 μl of a 250 ng/ml solution of abiotinylated polyclonal anti-IL6R antibody in PBS containing 1% casein(R&D systems, Article No. BAF227). After incubation for 30 minutes (RT),plates were washed three times (PBS containing 0.1% Tween20) andincubated for 30 minutes (RT) with 100 μl of a 1/5000 dilution (in PBScontaining 1% casein) of streptavidine conjugated with horseradishperoxidase (DaktoCytomation, Article No. P0397). After 30 minutes,plates were washed three times (PBS containing 0.1% Tween20) andincubated with 100 μl of slow TMB (Pierce, Article No. 34024). After 20minutes, the reaction was stopped with 100 μl HCl (1N). The absorbanceof each well was measured at 450 nm (Tecan Sunrise spectrophotometer),and corrected for absorbance at 620 nm. This assay measures freeNanobody as well as Nanobodies bound to sIL6R and/or cynomolgus serumalbumin. Concentration in each plasma sample was determined based on asigmoidal standard curve with variable slope of the respective Nanobody.The LLOQ and ULOQ of IL6R202 were 7.00 ng/ml and 300 ng/ml,respectively. The LLOQ and ULOQ of IL6R203 were 2.00 ng/ml and 70.0ng/ml, respectively.

Each individual plasma sample was analyzed in two independent assays andan average plasma concentration was calculated for pharmacokinetic dataanalysis.

Mean observed plasma concentration-time profiles (±SD) after intravenousadministration of IL6R04-HSA (2.0 mg/kg), IL6R202 or IL6R203 are shownin FIG. 41.

Basic pharmacokinetic parameters of IL6R04-HSA, IL6R202 and IL6R203after a single intravenous administration at 2.00 mg/kg in the male andfemale cynomolgus monkey are listed in Tables C-28, C-29 and C-30,respectively. All parameters were calculated with two-compartmentalmodeling, with elimination from the central compartment.

TABLE C-28 Basic pharmacokinetic parameters¹ of IL6R04-HSA after asingle intravenous administration at 2.00 mg/kg in the male and femaleCynomolgus Monkey. Monkey Monkey CV 1m 2f² Mean SD (%) C₍₀₎ (μg/ml) 50.649.0 49.8 ± 1.13 2.27 V_(ss) (mL/kg) 79.1 77.2 78.2 ± 1.34 1.72 V_(z)(mL/kg) 84.4 80.8 82.6 ± 2.55 3.08 V_(c) (mL/kg) 39.6 40.8 40.2 ± 0.8492.11 V_(t) (mL/kg) 39.5 36.4 38.0 ± 2.19 5.78 CL (mL/day/kg) 10.3 10.510.4 ± 0.141 1.36 CL_(d) (mL/day/kg) 41.6 55.7 48.7 ± 9.97 20.5 t_(1/2α)(day) 0.309 0.229 0.269 ± 0.0566 21.0 t_(1/2β) (day) 5.69 5.33 5.51 ±0.255 4.62 MRT (day) 7.70 7.37 7.54 0.233 3.10 AUC_(inf) (μg•day/ml) 194191 193 ± 2.12 1.10 AUC_(inf)/D (kg•day/ml) 0.0970 0.0955 0.0963 ±0.00106 1.10 ¹All parameters were calculated with two-compartmentalmodeling ²Estimate of PK parameters should be interpreted with cautionbecause the method of calculation ignores the immunological clearancedue to neutralizing antibodies.

TABLE C-29 Basic pharmacokinetic parameters¹ of IL6R202 after a singleintravenous administration at 2.00 mg/kg in the male and femaleCynomolgus Monkey. Monkey Monkey CV 3m 4f Mean SD (%) C₍₀₎ (μg/ml) 57.656.5 57.1 ± 0.778 1.36 V_(ss) (mL/kg) 65.1 60.6 62.9 ± 3.18 5.06 V_(z)(mL/kg) 70.0 64.6 67.3 ± 3.82 5.67 V_(c) (mL/kg) 34.7 35.4 35.1 ± 0.4951.41 V_(t) (mL/kg) 30.4 25.2 27.8 ± 3.68 13.2 CL 6.97 6.74 6.86 ± 0.1632.37 (mL/day/kg) CL_(d) 22.1 19.1 20.6 ± 2.12 10.3 (mL/day/kg) t_(1/2α)(day) 0.474 0.500 0.487 ± 0.0184 3.78 t_(1/2β) (day) 6.96 6.64 6.80 ±0.226 3.33 MRT (day) 9.35 8.99 9.17 0.255 2.78 AUC_(inf) 287 297 292 ±7.07 2.42 (μg•day/ml) AUC_(inf)/D 0.144 0.148 0.146 ± 0.00283 1.94(kg•day/ml) ¹All parameters were calculated with two-compartmentalmodeling

TABLE C-30 Basic pharmacokinetic parameters¹ of IL6R203 after a singleintravenous administration at 2.00 mg/kg in the male and femaleCynomolgus Monkey. Monkey Monkey CV 5m 6f Mean SD (%) C₍₀₎ (μg/ml) 67.187.8 77.5 ± 14.6 18.9 V_(ss) (mL/kg) 41.5 34.1 37.8 ± 5.23 13.8 V_(z)(mL/kg) 42.5 35.2 38.9 ± 5.16 13.3 V_(c) (mL/kg) 29.8 22.8 26.3 ± 4.9518.8 V_(t) (mL/kg) 11.7 11.3 11.5 ± 0.283 2.46 CL (mL/day/kg) 6.98 7.117.05 ± 0.0919 1.30 CL_(d) (mL/day/kg) 22.7 27.2 25.0 ± 3.18 12.8t_(1/2α) (day) 0.250 0.187 0.219 ± 0.0445 20.4 t_(1/2β) (day) 4.22 3.433.83 ± 0.559 14.6 MRT (day) 5.94 4.80 5.37 0.806 15.0 AUC_(inf)(μg•day/ml) 286 281 284 ± 3.54 1.25 AUC_(inf)/D (kg•day/ml) 0.143 0.1410.142 ± 0.00141 1.00 ¹All parameters were calculated withtwo-compartmental modeling

Example 28 Efficacy in Cynomolgus Monkey

In human and non-human primates, human IL-6 (hIL-6) has been reported toinduce acute phase protein synthesis (Gauldi et al, 1987; Asano et al.,1990). Acute phase proteins are defined as a set of plasma proteins,like C-reactive protein (CRP), serum amyloid A, haptoglobin, fibrinogen,albumin, transferrin and C3, increasing or decreasing concentrations byat least 25% in inflammatory disorders, mainly due to changes in theirproduction by hepatocytes (Gabay & Kushner, 1999). Patterns of cytokineproduction and acute phase response differ in different inflammatoryconditions. Therefore, acute phase changes reflect the presence andintensity of inflammation, making them diagnostically relevant.

In this study, the ability of IL6R202 and IL6R203 to block in vivo IL-6induced responses was examined in primates.

Adult cynomolgus monkeys (Macaca fascicularis), aged between 3 and 4years and weighing below 3 kg were housed individually in an environmentcontrolled room, at room temperature. The body weight of all individualswas recorded at first administration and weekly from there on. Theanimals were numbered and coupled in groups of 2 individuals (one maleand one female) with approximately equal mean body weight.

The lyophilized hIL-6 (Gentaur) was dissolved in 0.5 M acetic acid to afinal concentration of 100 μg/mL. One vial containing 500 μg IL-6 wasdissolved in 5 mL 0.5 M acetic acid. First, 3 mL of 0.5 M acetic acidwas added to the vial and the solution was swirled until alllyophilisate was dissolved. The solution was transferred to a sterilepolystyrene tube. The vial was rinsed with 2 mL of 0.5 M acetic acid andthis solution was transferred to the polystyrene tube as well, withsubsequent gentle mixing of the solution.

Human IL-6 was administered via an s.c. bolus injection of 1 mL/kg b.w.in 1% autologues Cynomolgus monkey heat inactivated serum in the dorsalregion.

8 groups (group 4 to 11, Table C-31) of animals received a single i.v.injection of IL6R202 or IL6R203, in one of four different dosages;namely 0.6, 2, 6 or 20 mg/kg b.w. in D-PBS. The Nanobodies® wereadministered via an i.v. bolus injection of 1 mL/kg b.w. in D-PBS intothe vena cephalica of the left or right arm. Subsequently after 1 hour,all animals were injected with the first of 7 daily injections s.c. ofhIL-6 (5 μg/kg b.w.). Two individuals of one group (group 12, 23m and24f, Table C-31) received no Nanobody® preadministration and served as anegative control.

Blood samples were collected prior to injection on test day 0 and fromthere on daily from test day 1 to test day 14 and on day 21.

The inflammation parameter CRP (serum C-reactive protein levels) is oneof the most important parameters for evaluating efficacy with respect toacute phase response in this animal model (animal model that was used inthe current study is described in detail in Asano et al. (1990) Blood 8,1602-1605). Furthermore, CRP is also one of the most importantnon-subjective parameters used in evaluating efficacy ofanti-inflammatory response compounds in human subjects as part ofclinical trials, which is well documented in the following references:Choy et al. (2002) Arthritis and Rheumatism 46 (12) 3143-50; Nishimotoet al. (2004) Arthritis and Rheumatism 50 (6) 1761-69; Maini et al.(2006) Arthritis and Rheumatism 54 (9) 2817-29. An increase in thelevels of CRP is one of the most relevant indications of inflammation.

In the current animal model, the CRP levels and a number of otherparameters were measured (results for the latter not shown); theinflammation parameter ESR (erythrocyte sedimentation rate) could not bedetermined in a quantitative manner (i.e. to provide a dose-responsecurve) using this model.

TABLE C-31 Chronologic schedule of hIL-6 and test item administrationAnimal Nanobody ® hIL6 Animal number Test Admin Admin group Male Femaleitem (day) dose (day) dose  4  7  8 IL6R202 1 0.6 1, 2, 3, 4, 5 μg/kgmg/kg 5, 6, 7 b.w. b.w.  5  9 10 IL6R202 1 2 1, 2, 3, 4, 5 μg/kg mg/kg5, 6, 7 b.w. b.w.  6 11 12 IL6R202 1 6 1, 2, 3, 4, 5 μg/kg mg/kg 5, 6, 7b.w. b.w.  7 13 14 IL6R202 1 20 1, 2, 3, 4, 5 μg/kg mg/kg 5, 6, 7 b.w.b.w.  8 15 16 IL6R203 1 0.6 1, 2, 3, 4, 5 μg/kg mg/kg 5, 6, 7 b.w. b.w. 9 17 18 IL6R203 1 2 1, 2, 3, 4, 5 μg/kg mg/kg 5, 6, 7 b.w. b.w. 10 1920 IL6R203 1 6 1, 2, 3, 4, 5 μg/kg mg/kg 5, 6, 7 b.w. b.w. 11 21 22IL6R203 1 20 1, 2, 3, 4, 5 μg/kg mg/kg 5, 6, 7 b.w. b.w. 12 23 24 — — —1, 2, 3, 4, 5 μg/kg 5, 6, 7 b.w.

Data on the level of CRP are presented in FIG. 42. At test day 2, theserum concentration of CRP in all groups, control and test, reached amaximum of up to 13 times the base level. A clear correlation betweenthe administered dosage of either Nanobody® and the level of increase inCRP was observed.

Example 29 Competition ELISA with Reference IgG

To analyze epitope specificity of a panel of Nanobodies in comparisonwith the Reference IgG, an ELISA experiment was designed where theReference IgG MRA was coated, and a dilution series of Nanobodiespre-incubated with a constant amount of IL6R was subsequentlytransferred to the wells.

Detection was performed using biotinylated anti-IL6R Mab M182(Pharmingen) and streptavidin-HRP. Data indicate that only for ReferenceFab, IL6R-03, IL6R65, IL6R13 and IL6R88 a competitive binding to theReference IgG was observed.

Data are presented in FIGS. 43, 44 and 45.

Example 30 Biacore Reference-Fab

To analyze epitope specificity of a panel of Nanobodies in comparisonwith the Reference-Fab, a Biacore experiment was designed where BN12, anon-neutralizing anti-human IL6R Mab (Diaclone) was coupled to the chipin Sodium acetate pH=4. Next, 100 nM IL-6R was injected on the chip.Then, 100 nM of an anti-IL-6R nanobody was injected. Finally, a mixtureof 100 nM Reference-Fab and 100 nM Nanobody was injected.

Results (FIG. 46) showed that, for all Nanobodies tested, theReference-Fab was still binding to IL-6R when IL-6R was already bound bythe Nanobodies. This indicated that the anti-IL-6R Nanobodies possiblyrecognized a different epitope on IL-6R. Only for Nanobodies IL6R-03,IL6R65, IL6R06, IL6R13 and IL6R88 a reduced binding was observed.

Example 31 Potency to Inhibit Binding of Human IL6 to Human, Rhesus andCynomolgus Monkey Soluble IL6R from Plasma

Potency to inhibit the binding of human IL6 to human, rhesus andcynomolgus monkey soluble IL6R present in plasma was analyzed for apanel of Nanobodies. A non-neutralizing anti-IL6R Mab BN12 (Diaclone)was coated on a microtiter well plate. Subsequently, plasma from human,rhesus or cynomolgus monkey pre-incubated with human IL6 and a dilutionserie of Nanobody was applied to the plate. Detection was performedusing anti-IL6-biotine followed by streptavidin-HRP reagent. Data arepresented in FIGS. 47, 48, 49 and 50.

Reference Example 1 Preparation of Reference Fab and IgG According to EP0 628 639 (=WO 92/19759)

Two representative anti-human IL-6R immunoglobulins according to EP 0628 639 (a Fab fragment and a full-sized IgG) were generated and used asreference compounds herein.

The Fab fragment and full-sized IgG were constructed based on theL-chain called “RV_(L)a” (see EP 0 628 639 B1, Table 2, version (a)) andthe H-chain called “RV_(H)f” (see EP 0 628 639 B1, Table 3, version(f)). These particular L-chain and H-chain were chosen for the purposesof constructing the reference compounds because, according to EP 0 268639 B1 (see for example paragraph [0074]), a reshaped human antibodycomprising said L-chain and said H-chain exhibited an ability to bind tohuman IL-6R at the same level as PM1, a mouse monoclonal antibodyagainst human IL-6R (see again EP 0 628 639 B1, paragraph [009] and thefurther references cited therein).

The full-length reference IgG consisted of the amino acid sequences ofSEQ ID NO: 629 (heavy chain) and SEQ ID NO: 630 (light chain). The Fabfragment consisted of the amino acid sequences of SEQ ID NO: 631 (heavychain regions V_(L)b and V_(H)f fused to the CH1 region of human IgG1)and SEQ ID NO: 632 (reshaped human PM-1 variable light chain fused tohuman Ckappa).

Encoding DNA fragments were generated by assembly PCR using partiallyoverlapping oligonucleotides. PCR products were cloned into a single,bi-cistronic vector which enables expression of functional,disulphide-linked Fab fragments in the periplasm of E. coli. Full-lengthIgG was produced in CHO cells transfected with 2 expression vectorscontaining the genes for the light and heavy chains. The gene encodingthe heavy chain was created by fusing V_(H)f to the constant region ofhuman IgG1. The light chain was as described in EP 0 268 639.

TABLE B-1 Nanobodies and polypeptides against the IL-6 receptor.“(t)” refers to a translated protein. Leader sequences and N-terminalsequences are given as SEQ ID NO's: 472-477. Nanobodies against the IL-6receptor <  > PMP40C9(t), SEQ ID NO: 399; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSAGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSS <  > PMP34F8(t), SEQ ID NO: 400; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSDGTTNTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTASYYCAADGHLNWGQPYVPCSQISWRGWNDYWGQGTQVTVSS <  > PMP34E9(t), SEQ ID NO: 401; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCATDRSVYYCSGDAPEEYYWGQGTQVTVSS <  > PMP34D2(t), SEQ ID NO: 402; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYFAIGWFRQAPGKERERVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRSVYYCSGGAPEEYYWGQGTQVTVSS <  > PMP34C3(t), SEQ ID NO: 403; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCVDSAPYDYWGRGTQVTVSS <  > PMP34A5(t), SEQ ID NO: 404; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLGYFAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCATDRSVYYCSGGAPEEYYWGQGTQVTVSS <  > PMP33G3(t), SEQ ID NO: 405; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLGYFAIGWFRQAPGKEREGVSCISSSDGSAYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCATDRSVYYCSGGAPEEYYWGQGTQVTVSS <  > PMP33C10(t), SEQ ID NO: 406; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGGSTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSS <  > PMP33A2(t), SEQ ID NO: 407; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSGGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSS <  > PMP32H5(t), SEQ ID NO: 408; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP32F10(t), SEQ ID NO: 409; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGISCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWILDGSPEFFKFWGQGTQVTVSS <  > PMP31F4(t), SEQ ID NO: 410; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP31D2(t), SEQ ID NO: 411; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSNWYLDGSPEFFKFWGQGTQVTVSS <  > PMP31C8(t), SEQ ID NO: 412; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP31C5(t), SEQ ID NO: 413; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISSDNAKNTVYLLMNSLKPEDTAVYYCAAEPPDSMWSLDGSPEFFKFWGQGTQVTVSS <  > PMP31B4(t), SEQ ID NO: 414; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP31B11(t), SEQ ID NO: 415; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP30G11(t), SEQ ID NO: 416; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCVDSAPYDYWGQGTQVTVSS <  > PMP30B6(t), SEQ ID NO: 417; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREAVACISSSDRSTYYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCAADLLRTPEFCSDSAPYDYWGQGTQVTVSS <  > PMP30B1(t), SEQ ID NO: 418; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWWRQAPGKGREGVSCISSGDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRSVYYCSGGAPEEYYWGQGTQVTVSS <  > PMP30A2(t), SEQ ID NO: 419; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCIGSSDDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCTDSAPYDYWGQGTQVTVSS <  > PMP30A10(t), SEQ ID NO: 420; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGGSTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSS <  > PMP28H6(t), SEQ ID NO: 421; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP28F7(t), SEQ ID NO: 422; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP28D4(t), SEQ ID NO: 423; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP28C7(t), SEQ ID NO: 424; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGDNTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  > PMP28B1(t), SEQ ID NO: 425; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLNYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTFYLQMNSLKPEDTAVYYCAAEGLGDSDSPCGAAWYNDYWGQGTQVTVSS <  > PMP28A2(t), SEQ ID NO: 426; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  >PMP40H5, SEQ ID NO: 427; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSS <  >PMP35H4, SEQ ID NO: 428; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSS <  >PMP35F4, SEQ ID NO: 429; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAIITWNSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAQYGLGYAEDY WGQGTQVTVSS<  >PMP35E11, SEQ ID NO: 430; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEHEGVSCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAERDVPARSLCGSYYWYDYRGQGTQVTVSS <  >PMP35C10, SEQ ID NO: 431; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVIHWSSGSTYYADPVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAFLPGPEGFHD YWGQGTQVTVSS<  >PMP34G9, SEQ ID NO: 432; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYW GQGTQVTVSS<  >PMP34G3, SEQ ID NO: 433; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERERVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCATDRSVYYCSGGAPEEYYWGQGTQVTVSS <  >PMP34E10, SEQ ID NO: 434; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWGRQAPGKEREFVATISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLAEFKYSD YADYWGQGTQVTVSS<  >PMP34C11, SEQ ID NO: 435; PRT; ->EVQLVESGGGLVQPGGSLRLSCAAAGFTLDYSAIGWFRQAPGKEREMFSCISGSDGSTWYADSVAGRFTISFDNAKNTVYLQMNSLKPEDTGLYICAVTGGVRGPCAYE YEYWGQGTQVTVSS<  >PMP34A12, SEQ ID NO: 436; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCVDSAPYDYWGQGTQVTVSS <  >PMP33A3, SEQ ID NO: 437; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYGAIGWFRQAPGKEREGVSCISSSTGSTYYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCAADKMWSPCLVAANEEALFEYDYWGQGTQVTVSS <  >PMP32E2, SEQ ID NO: 438; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGNIFDDNTMGWTWNRQPPGKQRELVAIIATDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLFSLRLGRDY WGQGTQVTVSS<  >PMP32E10, SEQ ID NO: 439; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSS <  >PMP32C9, SEQ ID NO: 440; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSS <  >PMP31A4, SEQ ID NO: 441; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGR DYWGQGTQVTVSS<  >PMP30C11, SEQ ID NO: 442; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVISRSGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCKAEVVAGDYDYW GQGTQVTVSS<  >PMP28G3, SEQ ID NO: 443; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGNIFSTETMGWYRQPPGKQRDVVATITHGGTTNYADSVKGRFTISRDNRKNTVYLQMNSLKPEDTGVYYCNARSSWYSPEYW GQGTQVTVSS<  >PMP28E11, SEQ ID NO: 444; PRT; ->EVQLVESGGGFVQAGGSLRLSCIASGDNFSINRMGWYRQALGKQRELVAIITNHGSTNYADAVKGRFTISRDYAKNTVYLQMNGLKPDDTAVYYCNAYISEVGTWRDD YWGQGIQVTVSS<  > 059B.IL6-R.cl5.7(t), SEQ ID NO: 445; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSGADAGWNRQTPGKEREFVAAINWSGNSTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCHAFRDDYYSEG KGTLVTVSS<  > 059A.IL6-Rcl4(t), SEQ ID NO: 446; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTLSSYDMGWYRQGPGKEREFVAAISWSGGGTDYVDSVKGRFTISRDTAKNTMYLQMNSLKPEDTAIYYCNALGTTDSDYEG ELYWGQGTQVTVSS<  > 059A.IL6-Rcl3(t), SEQ ID NO: 447; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDSYAIGWFRQAPGKEPEGVSCISTSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTADGGPHAPLTVQDMCVMAIADYWGQGTQVTVSS <  > 059A.IL6-Rcl2(t), SEQ ID NO: 448; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSNIAMGWIREAPGKEREFVAALTWSGGSTYYADSVKGRFTISRDSAKNTVYLQMNKLKPEDTAVYYCVADEEIHLIVSISIADFWGQGTQVTVSS <  > 059A.IL6-Rcl1(t), SEQ ID NO: 449; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGLTDDDFAIGWFRQAPGKEPEGVSCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYFCTALFDRCGSTWYY GMDYWGKGTLVTVSSBispecific polypeptides against the IL6 receptor<  >063.IL-6RPMP35C10cl8, SEQ ID NO: 450; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVIHWSSGSTYYADPVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAFLPGPEGFHDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6RPMP34G9cl2, SEQ ID NO: 451; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6RPMP34G3cl4-5, SEQ ID NO: 452; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKERERVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCATDRSVYYCSGGAPEEYYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6RPMP34A12cl2, SEQ ID NO: 453;PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCVDSAPYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6RPMP31A4cl4, SEQ ID NO: 454;PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP40H5cl4, SEQ ID NO: 455; PRT;-> EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP35F4cl3, SEQ IDNO: 456; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAIITWNSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAQYGLGYAEDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP35E11cl1, SEQ ID NO: 457; PRT;-> EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEHEGVSCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAERDVPARSLCGSYYWYDYRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP34E10cl3, SEQ ID NO: 458;PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWGRQAPGKEREFVATISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLAEFKYSDYADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP34C11cl3, SEQ ID NO: 459;PRT; -> EVQLVESGGGLVQPGGSLRLSCAAAGFTLDYSAIGWFRQAPGKEREMFSCISGSDGSTWYADSVAGRFTISFDNAKNTVYLQMNSLKPEDTGLYICAVTGGVRGPCAYEYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP33A3cl1, SEQ ID NO: 460; PRT;-> EVQLVESGGGLVQPGGSLRLSCAASGFTLDYGAIGWFRQAPGKEREGVSCISSSTGSTYYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCAADKMWSPCLVAANEEALFEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP32E2cl4, SEQ ID NO:461; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGNIFDDNTMGWTWNRQPPGKQRELVAIIATDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLFSLRLGRDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP32E10cl1, SEQ ID NO: 462; PRT;-> EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP32C9cl2, SEQ ID NO:463; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP30C11cl2, SEQ ID NO: 464;PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVISRSGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCKAEVVAGDYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP28G3cl3, SEQ ID NO: 465; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGNIFSTETMGWYRQPPGKQRDVVATITHGGTTNYADSVKGRFTISRDNRKNTVYLQMNSLKPEDTGVYYCNARSSWYSPEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  >063.IL-6R.PMP28E11cl4, SEQ ID NO: 466; PRT; ->EVQLVESGGGFVQAGGSLRLSCIASGDNFSINRMGWYRQALGKQRELVAIITNHGSTNYADAVKGRFTISRDYAKNTVYLQMNGLKPDDTAVYYCNAYISEVGTWRDDYWGQGIQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  > 059B.IL6-R.cl5.7(t), SEQ ID NO: 467; PRT;-> EVQLVESGGGLVQAGGSLRLSCAASGRTFSGADAGWNRQTPGKEREFVAAINWSGNSTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCHAFRDDYYSEGKGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  > 059A.IL6-Rcl4(t), SEQ ID NO: 468; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTLSSYDMGWYRQGPGKEREFVAAISWSGGGTDYVDSVKGRFTISRDTAKNTMYLQMNSLKPEDTAIYYCNALGTTDSDYEGELYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  > 059A.IL6-Rcl3(t), SEQ ID NO: 469; PRT;-> EVQLVESGGGLVQPGGSLRLSCAASGFTLDSYAIGWFRQAPGKEPEGVSCISTSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTADGGPHAPLTVQDMCVMAIADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  > 059A.IL6-Rcl2(t), SEQ ID NO: 470;PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSNIAMGWIREAPGKEREFVAALTWSGGSTYYADSVKGRFTISRDSAKNTVYLQMNKLKPEDTAVYYCVADEEIHLIVSISIADFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  > 059A.IL6-Rcl1(t), SEQ ID NO: 471; PRT;-> EVQLVESGGGLVQAGGSLRLSCAASGLTDDDFAIGWFRQAPGKEPEGVSCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYFCTALFDRCGSTWYYGMDYWGKGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Leader sequences and N-terminal sequences<  > llama leader 1, SEQ ID NO: 472; PRT; -> VKKLLFAIPLVVPFYAAQPAMA<  > llama leader 2, SEQ ID NO: 473; PRT; -> VKKLLFAIPLVVPFYAAQPAIA<  > llama leader 3, SEQ ID NO: 474; PRT; -> FELASVAQA <  > leadersequence, SEQ ID NO: 475; PRT; -> MKKTAIAIAVALAGLATVAQA <  > leadersequence, SEQ ID NO: 476; PRT; -> MKKTAIAFAVALAGLATVAQA <  > N-terminalsequence, SEQ ID NO: 477; PRT; -> AAAEQKLISEEDLNGAAHHHHHH** Trivalentbispecific polypeptides directed against the IL-6 receptor and humanserum albumin <  IL6R43, SEQ ID NO: 478“; PRT; ->”EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSS <  IL6R44, SEQ ID NO: 479“;PRT; ->” EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSS <  IL6R49, SEQ IDNO: 480“; PRT; ->” EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSS <  IL6R53, SEQ ID NO: 481“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSS <  IL6R54, SEQ IDNO: 482“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGW NDYWGQGTQVTVSS<  , SEQ ID NO: 483“; PRT; ->”AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSS <  , SEQ ID NO: 484“; PRT;->” AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSS <  , SEQ ID NO:485“; PRT; ->” AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSS <  , SEQ ID NO: 486“; PRT; ->”AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSS <  , SEQ ID NO:487“; PRT; ->” AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWR GWNDYWGQGTQVTVSS<  , SEQ ID NO: 488“; PRT; ->”EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  , SEQ ID NO: 489“; PRT;->” EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  , SEQ ID NO:490“; PRT; ->” EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  , SEQ ID NO: 491“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <  , SEQ IDNO: 492“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS Humanized trivalent bispecific polypeptides directedagainst the IL-6 receptor and human serum albumin <  , SEQ ID NO: 493“;PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 494“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 495“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 496“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS <  IL6R67, SEQ ID NO: 497“;PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 498“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO:499“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO:500“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO:501“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  IL6R203, SEQ IDNO: 502“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO:503“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:504“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:505“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:506“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:507“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:508“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:509“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R92, SEQ IDNO: 510“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:511“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:512“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:513“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:514“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:515“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 516“; PRT;->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 517“; PRT;->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 518“; PRT;->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 519“; PRT;->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 520“; PRT;->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:521“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:522“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:523“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:524“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:525“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:526“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:527“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:528“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:529“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:530“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:531“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:532“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:533“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:534“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:535“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:536“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO:537“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 538“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 539“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 540“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 541“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 542“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO:543“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO: 544“;PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO:545“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO:546“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO: 547“;PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:548“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:549“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:550“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:551“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:552“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:553“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:554“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:555“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:556“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:557“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO:558“; PRT; ->” EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS Humanizedbispecific polypeptides against the IL6 receptor and human serum albumin<  , SEQ ID NO: 559“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 560“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 561“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 562“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  IL6R66, SEQ ID NO: 563“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 564“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 565“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 566“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 567“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  IL6R202, SEQ ID NO: 568“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 569“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 570“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 571“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 572“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 573“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 574“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 575“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  IL6R91, SEQ ID NO: 576“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 577“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 578“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 579“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 580“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS <  , SEQ ID NO: 581“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 582“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 583“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 584“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 585“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSS <  , SEQ ID NO: 586“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO: 587“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO: 588“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO: 589“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO: 590“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  , SEQ ID NO: 591“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 592“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 593“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 594“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 595“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 596“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 597“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 598“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 599“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 600“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 601“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  , SEQ ID NO: 602“; PRT; ->”EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS Anti-IL-6 receptor Nanobody-human serumalbumin fusion proteins <  HSA-(GGS)4GG-IL6R04, SEQ ID NO: 603“; PRT;->” DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSGGSGGSGGSGGEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSS <  IL6R04-HSA,SEQ ID NO: 604“; PRT; ->”EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL <  IL6R201-HSA, SEQ ID NO:605“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL <  IL6R201-9GS-IL6R201-HSA, SEQ IDNO: 606“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQ AALGL<  IL6R201-20GS-IL6R201-HSA, SEQ ID NO: 607“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA EEGKKLV<  IL6R201-AAA-IL6R201-HSA, SEQ ID NO: 608“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL <  IL6R61, SEQ IDNO: 609“; PRT; ->” EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGR DYWGQGTLVTVSS<  IL6R62, SEQ ID NO: 610“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGR DYWGQGTLVTVSS<  IL6R63, SEQ ID NO: 611“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGR DYWGQGTLVTVSS<  IL6R64, SEQ ID NO: 612“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGR DYWGQGTLVTVSS<  IL6R65, SEQ ID NO: 613“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGR DYWGQGTLVTVSS<  IL6R71, SEQ ID NO: 614“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  IL6R72, SEQ ID NO: 615“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  IL6R73, SEQ ID NO: 616“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  IL6R74, SEQ ID NO: 617“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  IL6R75, SEQ ID NO: 618“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSS <  IL6R81, SEQ ID NO: 619“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R82, SEQ ID NO: 620“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R83, SEQ ID NO: 621“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R84, SEQ ID NO: 622“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R85, SEQ ID NO: 623“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R86, SEQ ID NO: 624“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R87, SEQ ID NO: 625“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R88, SEQ ID NO: 626“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R89, SEQ ID NO: 627“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  IL6R90, SEQ ID NO: 628“; PRT; ->”EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS <  REFERENCE IGG HEAVY CHAIN, SEQ ID NO: 629“; PRT;->” QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK <  REFERENCE IGGLIGHT CHAIN, SEQ ID NO: 630“; PRT; ->”DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC <  REFERENCE FAB HEAVYCHAIN, SEQ ID NO: 631“; PRT; ->”QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC <  REFERENCE FABLIGHT CHAIN, SEQ ID NO: 632“; PRT; ->”DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC <  IL6R MACACAFASCICULARIS, SEQ ID NO: 633“; PRT; ->”LAPGGCPAQEVARGVLTSLPGDSVTLTCPGGEPEDNATVHWVLRKPAEGSHLSRWAGVGRRLLLRSVQLHDSGNYSCYRAGRPAATVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSPTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKLSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPTQAPTTNKDDDNILSGDSANATSLP VQD

The invention claimed is:
 1. A method for the treatment of sepsis,cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangialproliferative glomerulonephritis, Kaposi's sarcoma, AIDS-relatedlymphoma, and inflammatory diseases, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of: at least one polypeptide that specifically binds tointerleukin-6 receptor (IL-6R) and that modulates the interactionbetween interleukin-6 (IL-6) and IL-6R, wherein the polypeptidecomprises 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3), in which: CDR1 ischosen from the group consisting of: a) the amino acid sequence of SEQID NO: 135; and b) an amino acid sequence having 3, 2, or 1 amino aciddifference with SEQ ID NO: 135 and that is derived from SEQ ID NO: 135by means of affinity maturation; and CDR2 is chosen from the groupconsisting of: c) the amino acid sequence of SEQ ID NO: 237; and d) anamino acid sequences having 3, 2, or 1 amino acid difference with SEQ IDNO: 237 and that is derived from SEQ ID NO: 237 by means of affinitymaturation; and CDR3 is chosen from the group consisting of: e) theamino acid sequence of SEQ ID NO: 339; and f) an amino acid sequenceshaving 3, 2, or 1 amino acid difference with SEQ ID NO: 339 and that isderived from SEQ ID NO: 339 by means of affinity maturation; and one ormore further pharmaceutically active compounds.
 2. The method accordingto claim 1, in which the CDR sequences of the polypeptide have at least80% amino acid identity, at least 90% amino acid identity, 95% aminoacid identity or more, or even essentially 100% amino acid identity withthe CDR sequence of SEQ ID NO:
 441. 3. The method acid according toclaim 1, wherein the polypeptide essentially consists of a domainantibody, of a single domain antibody, of a dAb, of a VHH, of ahumanized VHH, or of a camelized VH.
 4. The method according to claim 1,wherein the polypeptide essentially consists of a VHH, of a humanizedVHH, or of a camelized VH that a) has 80% amino acid identity with atleast one of the amino acid sequences of SEQ ID NOs: 399 to 471, inwhich for the purposes of determining the degree of amino acid identity,the amino acid residues that form the CDR sequences are disregarded; andin which: b) one or more of the amino acid residues at positions 11, 37,44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numberingare chosen from; at position 11: L, M, S, V, or W, at position 37: F, Y,H, I, L, or V, at position 44: G, E, A, D, Q, R, S, or L, at position45: L, R, C, I, L, P, Q, or V, at position 47: W, L, F, A, G, I, M, R,S, V, or Y, at position 83: R, K, N, E, G, I, M, Q, or T, at position84: P, A, L, R, S, T, D, or V, at position 103: W, P, R, or S, atposition 104: G, or D, and at position 108: Q, L, or R.
 5. The methodaccording to claim 1, wherein the polypeptide essentially consists of ahumanized VHH.
 6. The method according to claim 1, wherein thepolypeptide is chosen from the group consisting of: a) SEQ ID NO: 441;b) an amino acid sequence having at least 95% sequence identity with SEQID NO: 441; and c) an amino acid sequence having 3, 2 or only 1 aminoacid difference with SEQ ID NO:
 441. 7. The method according to claim 1,wherein the polypeptide further comprises one or more other groups,residues, moieties or binding units, optionally linked via one or morelinkers.
 8. The method according to claim 7, wherein the linker isselected from the group consisting of SEQ ID NOs: 37-41.
 9. The methodacid according to claim 7, wherein the one or more other groups,residues, moieties or binding units provide the polypeptide withincreased half-life.
 10. The method according to claim 9, wherein theone or more other groups, residues, moieties or binding units thatprovide the polypeptide with increased half-life are chosen from thegroup consisting of serum proteins or fragments thereof, binding unitsthat can bind to serum proteins, an Fc portion, and small proteins orpeptides that can bind to serum proteins.
 11. The method according toclaim 9, wherein the one or more other groups, residues, moieties orbinding units that provide the compound or construct with increasedhalf-life are chosen from the group consisting of domain antibodies,single domain antibodies, dAbs, VHHs, humanized VHHs, and VHs that canbind to serum albumin, human serum albumin, a serum immunoglobulin, orIgG.
 12. The method according to claim 11, wherein the one or more otherVHHs, humanized VHHs and VHs are selected from the group consisting ofSEQ ID NOs: 32-34.
 13. The method according to claim 1, wherein thecancer is chosen from the group consisting of multiple myeloma disease(MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma,B-lymphoproliferative disorder (BLPD), and prostate cancer.
 14. Themethod according to claim 1, wherein the inflammatory diseases arechosen from the group consisting of rheumatoid arthritis, systemic onsetjuvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease,ulcerative colitis, systemic lupus erythematosus (SLE), multiplesclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma,allergic asthma and autoimmune insulin-dependent diabetes mellitus. 15.The method according to claim 1, wherein the polypeptide is administeredintravenously or subcutaneously.
 16. The method according to claim 1,wherein the one or more further pharmaceutically active compound is usedfor treatment of an inflammatory disease chosen from the groupconsisting of rheumatoid arthritis, systemic onset juvenile idiopathicarthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma andautoimmune insulin-dependent diabetes mellitus.
 17. The method accordingto claim 1, wherein the at least one polypeptide and the one or morefurther pharmaceutically active compound are administered via adifferent route of administration.
 18. The method according to claim 1,wherein the at least one polypeptide and the one or more furtherpharmaceutically active compound are administered at different times.